Lactase can target cellular differentiation of Acanthamoeba castellanii belonging to the T4 genotype.

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.

Advances in Contact Lens Care Solutions: PVP-I Disinfectant and HAD Wetting Agents From Japan.

ABSTRACT: Half of the individuals who wear contact lenses use reusable lenses that require proper care. Improper contact lens (CL) care and using inadequate disinfecting solutions can lead to lens contamination, CL-related microbial keratitis, and Acanthamoeba keratitis. Oxidative disinfecting solutions, such as hydrogen peroxide, show higher efficacy than multipurpose solutions. Povidone-iodine (PVP-I), an oxidative disinfectant used in ophthalmic surgery, has been proven to be safe and effective. The PVP-I system, a CL disinfecting solution developed in Japan, has demonstrated excellent antimicrobial and antiviral properties. Although CL discomfort does not have a risk of ocular disorders with poor visual prognosis, such as keratitis, CL discomfort can still lead to lens dropout and thus needs to be addressed. To mitigate CL discomfort, it is essential to use disinfecting solutions containing surfactants and wetting agents that improve wettability of the lens surface. A CL solution containing hyaluronic acid derivatives (HADs) as wetting agents that permanently adhere to the lens surface to improve wettability of the lens surface was developed in Japan. There is potential for HAD to be integrated into various solutions. This article reviews the efficacy of novel PVP-I-based disinfecting solution and HAD wetting agents.

Antiamoebic properties of Methyltrioctylammonium chloride based deep eutectic solvents.

PURPOSE: This aim of this study was to assess anti-parasitic properties of deep eutectic solvents against eye pathogen, Acanthamoeba, often associated with the use of contact lens. METHODS: Assays were performed to investigate the effects of various Methyltrioctylammonium chloride-based deep eutectic solvents on Acanthamoeba castellanii, comprising amoebicidal assays, encystment assays, excystment assays, cytotoxicity assays by measuring lactate dehydrogenase release from human cells, and cytopathogenicity assays to determine parasite-mediated host cell death. RESULTS: In a 2 h incubation period, DES-B, DES-C, DES-D, and DES-E exhibited up to 85 % amoebicidal activity at micromolar doses, which was enhanced further following 24 h incubation. When tested in encystment assays, selected deep eutectic solvents abolished cyst formation and were able to block excystment of A. castellanii. All solvents exhibited minimal human cell cytotoxicity except DES-D. Finally, all tested deep eutectic solvents inhibited amoeba-mediated cytopathogenicity, except DES-B. CONCLUSIONS: Deep eutectic solvents show potent antiamoebic effects. These findings are promising and could lead to the development of novel contact lens disinfectants, as well as opening several avenues to explore the molecular mechanisms, various doses and incubation periods, and use of different bases against Acanthamoeba castellanii.

Anti-Acanthamoeba activity of a semi-synthetic mangostin derivative and its ability in removal of Acanthamoeba triangularis WU19001 on contact lens.

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.

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.

Differential Antimicrobial Efficacy of Preservative-Free Contact Lens Disinfection Systems against Common Ocular Pathogens.

Microbial keratitis is a devastating disease that can cause eye damage and blindness and can be the result of infections by several common ocular pathogens. Importantly, some of these pathogens, such as Acanthamoeba, are particularly unsusceptible to biocides in common contact lens care solutions. Therefore, the disinfection efficacy of preservative-free (PF) disinfection systems against bacteria, fungi, and Acanthamoeba trophozoites and cysts should be assessed as products with the most potential to be efficacious against resistant organisms. PF disinfection systems were analyzed for antimicrobial efficacy. These were the one-step (hydrogen peroxide-based) Clear Care and Clear Care Plus systems and the two-step (povidone-iodine-based) Cleadew system. Stand-alone challenges using bacteria, fungi, and Acanthamoeba were prepared according to the International Standards Organization method 14729. These same challenges were also conducted in the presence of the following contact lenses: Boston RGP, Acuvue Oasys, Biofinity, Ultra, and 2-week PremiO. All challenges were performed at the manufacturer's recommended disinfection time. All preservative-free disinfection systems demonstrated similarly high rates of antimicrobial efficacy when challenged with bacteria or fungi, with or without lenses. However, both Clear Care and Clear Care Plus demonstrated significantly greater disinfection efficacy against Acanthamoeba trophozoites and cysts, with and without lenses (P < 0.05). Cleadew efficacy was impacted by the addition of contact lenses, whereas Clear Care/Clear Care Plus maintained similar efficacies in the absence or presence of lenses. While both hydrogen peroxide and povidone-iodine are highly effective against bacteria and fungi, hydrogen peroxide maintains significantly greater disinfection capabilities than povidone-iodine against all forms of Acanthamoeba. IMPORTANCE Understanding the most efficacious products will allow clinicians to best communicate to patients and consumers the safest products on the market to reduce adverse events, including microbial keratitis, during contact lens use.

Amoebicidal Activity of Poly-Epsilon-Lysine Functionalized Hydrogels.

Purpose: To determine the amoebicidal activity of functionalized poly-epsilon-lysine hydrogels (pɛK+) against Acanthamoeba castellanii. Methods: A. castellanii trophozoites and cysts were grown in the presence of pɛK solution (0-2.17 mM), pɛK or pɛK+ hydrogels, or commercial hydrogel contact lens (CL) for 24 hours or 7 days in PBS or Peptone-Yeast-Glucose (PYG) media (nutrient-deplete or nutrient-replete cultures, respectively). Toxicity was determined using propidium iodide and imaged using fluorescence microscopy. Ex vivo porcine corneas were inoculated with A. castellanii trophozoites ± pɛK, pɛK+ hydrogels or commercial hydrogel CL for 7 days. Corneal infection was assessed by periodic acid-Schiff staining and histologic analysis. Regrowth of A. castellanii from hydrogel lenses and corneal discs at 7 days was assessed using microscopy and enumeration. Results: The toxicity of pɛK+ hydrogels resulted in the death of 98.52% or 83.31% of the trophozoites at 24 hours or 7 days, respectively. The toxicity of pɛK+ hydrogels resulted in the death of 70.59% or 82.32% of the cysts in PBS at 24 hours or 7 days, respectively. Cysts exposed to pɛK+ hydrogels in PYG medium resulted in 75.37% and 87.14% death at 24 hours and 7 days. Ex vivo corneas infected with trophozoites and incubated with pɛK+ hydrogels showed the absence of A. castellanii in the stroma, with no regrowth from corneas or pɛK+ hydrogel, compared with infected-only corneas and those incubated in presence of commercial hydrogel CL. Conclusions: pɛK+ hydrogels demonstrated pronounced amoebicidal and cysticidal activity against A. castellanii. pɛK+ hydrogels have the potential for use as CLs that could minimize the risk of CL-associated Acanthamoeba keratitis.

Cytotoxicities and wound healing effects of contact lens multipurpose solution on human corneal epithelial cell.

CLINICAL RELEVANCE: Contact lens multipurpose solutions (MPSs) contain several components that have the potential to cause corneal epithelial cell toxicity. Evaluating the components and the toxic effect of MPS should be considered for effective eye care. BACKGROUND: The cytotoxic and wound healing effects of five commercially available MPSs on human corneal epithelial cells (HCECs) are is investigated. METHODS: The following commercially available MPSs were used: Queen's PLURISOL®, Frenz®, Boston SIMPLUS®, DL+PLUS EYE® (DL), and NEW YORK DEFINE® (NY). The proliferation of HCECs exposed to each MPS for 1, 6, and 24 h and the cytotoxicity of these solutions were analyzed using methyl thiazolyl tetrazolium-based colorimetric and lactate dehydrogenase leakage assays, respectively. The cellular morphology was evaluated by inverted phase-contrast and electron microscopy. A scratch-wound assay was performed to measure wound widths 24 h after confluent HCEC monolayers were scratch-wounded. RESULTS: The tested MPS had a time-dependent inhibitory effect on HCEC proliferation and cytotoxicity, significantly at 24 h after exposure (p< 0.05 in all MPSs). HCECs exposed to MPS detached from the bottom of the culture dishes, showed degenerative changes such as loss of microvilli, cytoplasmic vacuole formation and nuclear condensation, and decreased wound healing, compared to the controls (p< 0.001 in Boston, DL and NY). Among the tested MPS, DL and NY were more cytotoxic and showed less wound healing. CONCLUSION: MPS has a toxic effect on HCECs, which is dependent on the concentration of the disinfecting component. Since the components that constitute the MPS are absorbed and retained in the lens, cautious scrutiny of the concentration and attention to lens cleaning are warranted to mitigate the related cytotoxicity.

Contact lenses contamination by Acanthamoeba spp. in Upper Egypt.

BACKGROUND: Acanthamoeba spp. are one of the free-living amoeba that spread worldwide causing keratitis. Owing to the increase in the use of lenses, whether for medical or cosmetic purposes, the incidence of disease increases every year. Contamination of the lenses with the Acanthamoeba trophozoites or cysts may lead to eye infection and cause sight-threatening keratitis in human. We isolated Acanthamoeba spp. from new lenses, used lenses, and contact lens disinfecting solutions and identified them based on morphological characteristics and molecular test. METHODS: New and used lenses and contact lens disinfecting solutions were cultured on monogenic media. Light and scanning electron microscope was used to identify Acanthamoeba spp. morphological features. Genotype identification was also evaluated using PCR sequencing of 18S rRNA gene specific primer pair JDP1 and JDP2. RESULTS: A hundred samples were examined, 29 (29%) were infected with Acanthamoeba spp. That belonged to two strains of Acanthamoeba (Acanthamoeba 41 and Acanthamoeba 68). 18S rRNA of the Acanthamoeba 41 had 99.69% sequence identity to Acanthamoeba castellanii clone HDU-JUMS-2, whereas Acanthamoeba 68 had 99.74% similar pattern to that of Acanthamoeba sp. isolate T4 clone ac2t4 that are morphologically identified as Acanthamoeba polyphaga. The obtained data revealed that the isolated strains belong to T4 genotype that was evolutionarily similar to strains isolated in Iran. CONCLUSIONS: Cosmetic lenses and disinfectant solutions are a major transmissible mode for infection. This genotype is common as the cause of Acanthamoeba keratitis. To avoid infection, care must be taken to clean the lenses and their preservative solutions and prevent contamination with the parasite.

Effect of Water Exposure on Contact Lens Storage Case Contamination in Soft Lens Wearers.

SIGNIFICANCE: Water exposure during contact lens wear can transfer pathogenic microorganisms to the eye, causing ocular complications. This study determined that water exposure is prevalent among lens wearers and is independently associated with higher case contamination. Contact lens compliance education to minimize water exposure should be reenforced by contact lens organizations and practitioners. PURPOSE: Given the increasing association between water exposure and contact lens disease, this study aimed to explore the effect of water exposure and storage case contamination in soft contact lens users. METHODS: A total of 165 daily soft lens wearers completed a self-administered questionnaire on contact lens hygiene. Lens cases were collected, and microbial analysis was conducted using an adenosine triphosphate assay (for overall microbial bioburden) and limulus amebocyte lysate assay (for endotoxin levels). The lens hygiene (excellent, >35; moderate, 28 to 35; poor, <28) and water contact (≤1, good; >1, poor) scores measured the compliance and water exposure during lens wear, respectively. Multiple regression determined the association between water behaviors and case contamination. RESULTS: The average (standard deviation) age of participants was 28 (13.5) years (18 to 78 years), and 65% were female. The average overall microbial bioburden of storage cases was 3.10 (0.98) log colony-forming unit/mL (1.09 to 5.94 log colony-forming unit/mL). Forty-five percent of participants reported showering, 49.7% reported swimming while wearing lenses (65.4% of whom swam without goggles), 27.8% used wet hands to handle lenses, and 17.5% used tap water to rinse storage cases. Showering with lenses significantly increased the risk of higher storage case bioburden (P = .001), whereas using wet hands (P = .01) doubled the risk of higher case endotoxin levels (odds ratio, 2.41; confidence interval 1.19 to 4.86). CONCLUSIONS: Water contact behavior while wearing lenses is prevalent among soft lens wearers and is associated with higher case contamination. Practitioners may improve contact lens education to reduce water exposure and case contamination to reduce the risk of lens-related adverse events.

Contact lens procurement and usage habits among adults in Sudan.

OBJECTIVE: The study aimed to assess contact lens (CL) wear and care habits among adults in Sudan. DESIGN: An observational, non-interventional, multi-center, cross-sectional study was conducted in the CL departments of all the eye hospitals and centers, and optical centers in the Khartoum State, Sudan. PARTICIPANTS: Established CL wearers residing in Khartoum State, Sudan. MAIN OUTCOMES AND MEASURES: CL wear profile, CL usage habits, hand and CL hygiene habits were assessed using an interviewer-administered questionnaire. RESULTS: The average age of the 442 participants was 24.57 (± 4.87) years. Women comprised 92% of the sample. Smoking was reported by 15.8% of the sample. 81.9% wore soft spherical CL, with 43.4% of the participants were prescribed CL on a yearly replacement schedule. Only 68.3% were prescribed CL by an eye care practitioner. Multipurpose solutions were used by 78.8% of the sample to clean CL, and by 52% to clean the lens case. Compliance rate for CL wear and care among participants was 81.1%. None of the participants reported sharing CL or CL case and rinsing the case with tap water. High compliance level was observed for overnight wear; swimming and showering with CL; handwashing before CL insertion; and cleaning of CL. Moderate compliance rates were recorded for cleaning and replacing lens case, and topping up or sharing solution. Low compliance was noted for attending after care visits. CONCLUSIONS AND RELEVANCE: CL wearers in Sudan exhibit high to moderate levels of compliance to most contact lens wear and care aspects, except for attending aftercare visits. CL practitioners in Sudan are encouraged to prescribe CL appropriate to the lifestyle and economic situation of patients and actively recommend care products. In addition, practitioners need to follow up with patients to attend aftercare appointments, where hygienic wear and care habits should be emphasized.

Gas-Permeable Contact Lenses and Water Exposure: Practices and Perceptions.

SIGNIFICANCE: This study provides insight into the current recommendations, clinical behaviors, and risk assessments of eye care professionals on the topic of rigid contact lens exposure to tap water. This knowledge may motivate professional organizations to develop initiatives to educate eye care professionals on the dangers of contact lens exposure to water. PURPOSE: The purpose of this study was to investigate the practice patterns and risk perceptions of eye care professionals regarding gas-permeable contact lens exposure to tap water. METHODS: A branched-logic survey was started by 320 clinicians, vision scientists, and industry personnel and was fully completed by 272 participants. The survey queried participants about their current practices, recommendations, and perceptions of risk regarding exposure of gas-permeable contact lenses to tap water. RESULTS: Of those who prescribe gas-permeable contact lenses, 57.4% reported rinsing lenses with tap water, whereas only 32.7% reported engaging in this same behavior in front of patients. Of those who reported never rinsing lenses with tap water, 85.6% indicated that rinsing lenses with water increases infection risk, whereas 52.1% of those who rinse lenses with water reported the same perceived risk (P < .001). Of all participants, 60% indicated that wearing contact lenses while showering increases infection risk, and 50.2% of all participants advised patients to avoid this behavior. CONCLUSIONS: A discrepancy exists between perceptions of risk concerning exposure of contact lenses to water and both clinical practices and patient education provided by professionals.

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.

Detección de Acanthamoeba por reacción en cadena de la polimerasa en tiempo real en líquidos de conservación de lentes de contacto / Detection of Acanthamoeba sp. by real-time polymerase chain reaction in contact lens solutions

RESUMEN Objetivo: Estandarizar una técnica de reacción en cadena de la polimerasa en tiempo real para la detección del parásito e identificar Acanthamoeba en líquidos conservantes de lentes de contacto. Métodos: Se realizó un estudio descriptivo observacional de corte transversal sobre la técnica de reacción en cadena de la polimerasa en tiempo real para la detección de Acanthamoeba, en el Instituto de Investigaciones en Ciencias de la Salud de la ciudad de Asunción, en Paraguay. Se analizaron 110 líquidos conservantes aportados por usuarios sanos de lentes de contacto, mediante reacción en cadena de la polimerasa en tiempo real y cultivo en medio PAGE - SDS. Resultados: Se estandarizó con éxito la técnica de reacción en cadena de la polimerasa en tiempo real con límite de sensibilidad de 1 pg/µL. Se aisló Acanthamoeba a partir de una muestra (1 por ciento) por método de cultivo, mientras que la carga parasitaria en el líquido conservante fue inferior al límite de detección de la reacción en cadena de la polimerasa en tiempo real. El ADN obtenido del cultivo de dicha muestra fue positivo para Acanthamoeba por este método. Conclusión: El sistema estandarizado presenta buena sensibilidad y podrá ser incorporado en los laboratorios que cuentan con acceso a equipos de reacción en cadena de la polimerasa en tiempo real para un diagnóstico rápido y más eficiente en casos de sospechas de queratitis amebiana. Recomendamos el uso combinado de métodos moleculares y cultivo para aumentar la potencia del diagnóstico, sobre todo en muestras donde la carga parasitaria es muy baja(AU)

ABSTRACT Objective: Standardize a real-time polymerase chain reaction technique for detection of the parasite and identify Acanthamoeba in contact lens solutions. Methods: A cross-sectional observational descriptive study was conducted about a real-time polymerase chain reaction technique for detection of Acanthamoeba at the Institute of Health Sciences Research in the city of Asunción, Paraguay. A total 110 solutions were analyzed, which were provided by healthy contact lens users, by real-time polymerase chain reaction and culture in SDS-PAGE medium. Results: Successful standardization was achieved of the real-time polymerase chain reaction technique with a sensitivity limit of 1 pg/µl. Acanthamoeba was isolated from one sample (1 percent) by culture, whereas the parasite load in the contact lens solution was below the detection limit of the real-time polymerase chain reaction technique. The DNA obtained from the culture of that sample was positive for Acanthamoeba by the real-time polymerase chain reaction technique method. Conclusion: The system standardized exhibits good sensitivity and may be incorporated into laboratories with real-time polymerase chain reaction technique equipment for a rapid and more efficient diagnosis of suspected amoebic keratitis. We recommend the combined use of molecular methods and culture to enhance diagnostic power, mainly in samples where the parasite load is very low(AU)

Microbial Contamination of Rigid Gas Permeable (RGP) Trial Lenses and Lens Cases in China.

Purpose: This study aimed to evaluate the microbial contamination level and its influencing factors of rigid gas permeable (RGP) trial lenses and lens cases in China.Materials and Methods: A total of 107 RGP trial lenses and lens cases were collected from 7 main hospitals or optometric centers in China. Three sites including the lenses, case interiors and case screw tops were sampled for bacterial and fungal culture and identification. The contamination rates of these three sites and their relationship with lens care regimes were further analyzed.Results: The overall contamination rate was 73.8% for either lenses or cases, and 43.0% of lenses, 57.0% of case interiors and 65.4% of case screw tops respectively. The most frequently isolated microorganisms were Serratia spp., Burkholderia spp., Pandoraea spp., and Achromobacter spp. from all three sites. The contamination rate was positively related to the lens use frequency. Compared with dry-stored lenses, the contamination rate was significantly higher in wet-stored group (P < .001*). Inadequate disinfection and improper lens and case care regimes were also associated with higher contamination rates.Conclusions: Our study reported that the RGP trial lenses and cases used for fittings had a considerably high contamination rate. The safe use of RGP trial lenses and education of optometrists on the regular maintenance of trial lenses should be emphasized.

Synthesis and in vitro activity of new biguanide-containing dendrimers on pathogenic isolates of Acanthamoeba polyphaga and Acanthamoeba griffini.

The genus Acanthamoeba can cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). The treatment of these illnesses is hampered by the existence of a resistance stage that many times causes infection relapses. In an attempt to add new agents to our chemotherapeutic arsenal against acanthamebiasis, two Acanthamoeba isolates were treated in vitro with newly synthesized biguanide dendrimers. Trophozoite viability analysis and ultrastructural studies showed that dendrimers prevent encystment by lysing the cellular membrane of the amoeba. Moreover, one of the dendrimers showed low toxicity when tested on mammalian cell cultures, which suggest that it might be eventually used as an amoebicidal drug or as a disinfection compound in contact lens solutions.

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.

Biocidal Efficacy of a Hydrogen Peroxide Lens Care Solution Incorporating a Novel Wetting Agent.

PURPOSE: To compare the antimicrobial effects of CLEAR CARE, a 3% hydrogen peroxide (H2O2) solution formulated for simultaneous cleaning, daily protein removal, disinfection, and storage of soft (hydrophilic) hydrogel, silicone hydrogel, and gas-permeable contact lenses, and CLEAR CARE PLUS, consisting of the 3% H2O2 solution plus a novel wetting agent, polyoxyethylene-polyoxybutylene (EOBO-21). METHODS: Three lots each of the 2 solutions were incubated with 5 compendial microorganisms required by the Food and Drug Administration (FDA) 510(k) and International Organization for Standardization (ISO) 14729 stand-alone procedures, 4 clinical isolates of Gram-positive and Gram-negative bacteria, and trophozoites and cysts of 2 Acanthamoeba strains that are associated with microbial keratitis. Microbial loads were evaluated after disinfection and neutralization. RESULTS: Both solutions exceeded the FDA/ISO stand-alone primary criteria against Gram-positive and Gram-negative compendial bacteria, yeast, and mold after only 1.5-hr disinfection/neutralization. At the recommended minimum disinfection time, bacteria were reduced by 4.4 to 5.1 logs, yeast by 4.4 to 4.9 logs, and mold by 2.9 to 3.5 logs with and without organic soil. In addition, both solutions eliminated or effectively reduced populations of clinically relevant ocular bacterial isolates (4.5-5.0 logs), Acanthamoeba trophozoites (3.4-4.2 logs), and cysts (1.5-2.1 logs). CONCLUSION: Both solutions eliminated or reduced populations of FDA/ISO compendial bacteria and fungi as well as clinically relevant microorganisms and Acanthamoeba trophozoites and cysts. The addition of EOBO-21 to the 3% H2O2 lens care solution had no impact on antimicrobial activity.

Effect of 2, 6-Dichlorobenzonitrile on Amoebicidal Activity of Multipurpose Contact Lens Disinfecting Solutions.

Multipurpose contact lens disinfecting solutions (MPDS) are widely used to cleanse and disinfect microorganisms. However, disinfection efficacy of these MPDS against Acanthamoeba cyst remain insufficient. 2, 6-dichlorobenzonitrile (DCB), a cellulose synthesis inhibitor, is capable of increasing the amoebical effect against Acanthamoeba by inhibiting its encystation. In this study, we investigated the possibility of DCB as a disinfecting agent to improve the amoebicidal activity of MPDS against Acanthamoeba cyst. Eight commercial MPDS (from a to h) were assessed, all of which displayed insufficient amoebicidal activity against the mature cysts. Solution e, f, and h showed strong amoebicidal effect on the immature cysts. Amoebicidal efficacy against mature cysts remained inadequate even when the 8 MPDS were combined with 100 µM DCB. However, 4 kinds of MPDS (solution d, e, f, and h) including 100 µM DCB demonstrated strong amoebicidal activity against the immature cysts. The amoebicidal activity of solution d was increased by addition of DCB. Cytotoxicity was absent in human corneal epithelial cells treated with either DCB or mixture of DCB with MPDS. These results suggested that DCB can enhance the amoebicical activity of MPDS against Acanthamoeba immature cyst in vitro.