In Vitro Amoebicidal Activity of Titanium Dioxide/UV-A Combination Against Acanthamoeba.

Purpose: To assess the amoebicidal effect of titanium dioxide (TiO2)/UV-A combination against Acanthamoeba sp trophozoites and cysts. Methods: The amoebicidal effect of the TiO2/UV-A combination was tested on trophozoites and cysts of clinical isolates of Acanthamoeba hatchetti and Acanthamoeba sp genotype T4, obtained from two severe cases of ulcerative keratitis. Samples of cultured Acanthamoeba were transferred to a 96-well plate. We tested the effect of sterile water (blank control), TiO2 alone, UV-A alone, TiO2 and additional UV-A exposure, chlorhexidine 0.02% alone, chlorhexidine 0.02% and TiO2, chlorhexidine and UV-A, chlorhexidine 0.02% and TiO2, and additional UV-A exposure. Cell viability assessment was done using the trypan blue dye exclusion method. Results: The combination of TiO2 with UV-A demonstrated antitrophozoite and anticyst activity (P < 0.05). This in vitro study showed a synergistic effect of the association of chlorhexidine with TiO2 and UV-A on cysts (P < 0.001). Conclusions: Given the in vitro synergistic effectiveness of the association of chlorhexidine with TiO2 and UV-A against cysts, the treatment of Acanthamoeba keratitis could be improved by this new therapeutic approach.

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.

Riboflavin and ultraviolet A as adjuvant treatment against Acanthamoeba cysts.

BACKGROUND: Experimental studies have shown that the standard dose of riboflavin (R) or R + ultraviolet-A (UVA) as solo treatment are not able to exterminate Acanthamoeba cysts or even trophozoites. The purpose of this study is to determine whether the application of R + UVA can enhance the cysticidal effects of cationic antiseptic agents in vitro. METHODS: The log of either polyhexamethylene biguanide or chlorhexidine minimal cysticidal concentration in solutions containing riboflavin (concentrations 0.1, 0.05 and 0.025%) plus either Acanthamoeba castellanii cysts or Acanthamoeba polyphaga cysts was determined and compared in groups treated with UVA 30 mW/cm(2) for 30 min and in control groups (with no exposure to UVA). A permutation test was used to determine the P value associated with treatment. RESULTS: Regardless of the riboflavin concentration and UVA treatment condition, no trophozoites were seen in plates where the cysts were previously exposed to cationic antiseptic agent concentrations ≥200 µg/mL for Acanthamoeba castellanii samples and ≥100 µg/mL for A. polyphaga samples. There was no statistical evidence that R + UVA treatment was associated with minimal cysticidal concentration (P = 0.82). CONCLUSION: R + UVA in doses up to 10 times higher than recommended for corneal crosslinking does not enhance the cysticidal effect of either polyhexamethylene biguanide or chlorhexidine in vitro.

The efficacy of Acanthamoeba cyst kill and effects upon contact lenses of a novel ultraviolet lens disinfection system.

PURPOSE: To assess the efficacy of a novel ultraviolet (UV) lens device on the killing of Acanthamoeba cysts and the impact of efficacious doses of UV upon soft contact lens parameter and material characteristics. DESIGN: Prospective, in vitro, experimental study of a device. METHODS: A UV lens device was constructed and used to expose Acanthamoeba cysts to various levels of UV irradiation. Once an efficacious dose, as defined by a greater than 3 log reduction, was determined (130 mJ/cm(2)), 6 soft contact lens materials (etafilcon A, senofilcon A, galyfilcon A, lotrafilcon A, polymacon, and comfilcon A) were exposed to that dose for 30 cycles and tested for visual parameters, mechanical parameters, and cytotoxicity. RESULTS: The UV device produced an average log reduction of over 3.5 log of Acanthamoeba cysts when the lens and solution inside of the inset case was irradiated with 130 mJ per cm(2) of UV or greater. After 30 cycles of 130 mJ per cm(2) UV dose each, no gross changes were observed in mechanical properties or cytotoxicity tests in any soft contact lenses tested. In visual parameters, polymacon and lotrafilcon A exhibited a shift in sphere power and diameter, respectively. CONCLUSIONS: The novel UV lens device was able to provide a marked log reduction to Acanthamoeba cysts, one of the most resistant ocular disease-causing organisms found in lens cases, without a detrimental effect on many lens materials.

Evaluation of in vitro efficacy of combined riboflavin and ultraviolet a for Acanthamoeba isolates.

PURPOSE: To evaluate in vitro the amoebicidal effects of riboflavin and ultraviolet A (UVA) collagen cross-linking. DESIGN: Experimental study, laboratory investigation. METHODS: Two different strains of Acanthamoeba species were tested identically. Four treatment groups were considered: group 1 consisted of 0.1% riboflavin and 30-minute UVA irradiation; group 2 consisted of 0.1% riboflavin and 60-minute UVA irradiation; group 3 consisted of no riboflavin and no UVA exposure; group 4 consisted of 0.1% riboflavin and no UVA exposure. The application of UVA was performed under the parameters used for in vivo corneal collagen cross-linking. RESULTS: In all cases, cysts and trophozoites were detected 24 hours after treatment at a radial distance from the center of the seeding point more than 5 mm, indicating that the amoebae were viable. All treated and untreated groups of amoebae from the 2 strains exhibited growth (radii of 14 to 15 mm in groups 1, 3, and 4; radius of 12 mm in group 2). The final morphologic features of the 2 strains of trophozoites that received treatment were similar to those of the initial seeding group and the untreated control group. CONCLUSIONS: The results obtained in our study show that a single dose (30 or 60 minutes) of cross-linking cannot achieve eradication in the 2 different Acanthamoeba strains examined. However, in vitro results do not always indicate in vivo efficacy, so future studies should test the validity of this treatment for Acanthamoeba keratitis.

Antimicrobial activity of simulated solar disinfection against bacterial, fungal, and protozoan pathogens and its enhancement by riboflavin.

Riboflavin significantly enhanced the efficacy of simulated solar disinfection (SODIS) at 150 watts per square meter (W m(-2)) against a variety of microorganisms, including Escherichia coli, Fusarium solani, Candida albicans, and Acanthamoeba polyphaga trophozoites (>3 to 4 log(10) after 2 to 6 h; P < 0.001). With A. polyphaga cysts, the kill (3.5 log(10) after 6 h) was obtained only in the presence of riboflavin and 250 W m(-2) irradiance.

Resistance of Acanthamoeba cysts to disinfection treatments used in health care settings.

Free-living amoebae that belong to the genus Acanthamoeba are widespread in the environment, including water. They are responsible for human infections and can host pathogenic microorganisms. Under unfavorable conditions, they form cysts with high levels of resistance to disinfection methods, thus potentially representing a threat to public health. In the present study we evaluated the efficacies of various biocides against trophozoites and cysts of several Acanthamoeba strains. We demonstrated that disinfectant efficacy varied depending on the strains tested, with environmental strains demonstrating greater resistance than collection strains. Trophozoites were inactivated by all treatments except those using glutaraldehyde as an active compound: for these treatments, we observed resistance even after 30 min exposure. Cysts resisted many treatments, including certain conditions with glutaraldehyde and other biocides. Moist heat at 55 degrees C was not efficient against cysts, whereas exposure at 65 degrees C was. Several chemical formulations containing peracetic acid, hydrogen peroxide, or ortho-phthalaldehyde presented greater efficacy than glutaraldehyde, as did ethanol and sodium hypochlorite; however, some of these treatments required relatively long incubation times to achieve cyst inactivation. Amoebal cysts can be highly resistant to some high-level disinfectants, which has implications for clinical practice. These results highlight the need to consider the effective disinfection of protozoa in their vegetative and resistant forms due to their intrinsic resistance. This is important not only to prevent the transmission of protozoa themselves but also due to the risks associated with a range of microbial pathogens that are found to be associated intracellularly with these microorganisms.

Mechanisms involved in the photosensitized inactivation of Acanthamoeba palestinensis trophozoites.

AIMS: To advance our understanding of the mechanisms involved in the RLP068 phthalocyanine-photosensitized inactivation of Acanthamoeba palestinensis trophozoites through a precise identification of the targets of the photoprocess in both the cytosolic and mitochondrial compartments. METHODS AND RESULTS: We followed the activities of selected marker enzymes as well as we performed fluorescence and transmission electron microscopy investigations of the alterations induced by the photoprocess in the fine structure of subcellular compartments. RLP068 is preferentially located in the contractile vacuole: the fluorescence in that site is particularly evident in the unirradiated cells and becomes more diffused after irradiation. Electron microscopic analysis of photosensitized A. palestinensis cells clearly shows that the swelling of trophozoites and the appearance of vacuoles spread throughout the cytoplasm after phototreatment. The activity of a typical cytoplasmic enzyme, such as lactate dehydrogenase, underwent a 35% decrease as a consequence of the photoprocess, reflecting the photodamage induced by migrating phthalocyanine molecules in their micro-environment. CONCLUSIONS: The presence of multiple targets for the phthalocyanine-photosensitized process is of utmost importance because this pattern of cell damage makes it unlikely that photoresistant A. palestinensis strains are gradually selected or mutagenic phenomena are developed as a consequence of the photoinduced damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Photosensitization via phthalocyanines appears to represent an efficient and safe approach for achieving a close control of the population of a potentially pathogenic protozoan such as A. palestinensis, opening new perspectives for the disinfection of microbiologically polluted waters.

Solar disinfection of poliovirus and Acanthamoeba polyphaga cysts in water - a laboratory study using simulated sunlight.

AIMS: To determine the efficacy of solar disinfection (SODIS) in disinfecting water contaminated with poliovirus and Acanthamoeba polyphaga cysts. METHODS AND RESULTS: Organisms were subjected to a simulated global solar irradiance of 850 Wm(-2) in water temperatures between 25 and 55 degrees C. SODIS at 25 degrees C totally inactivated poliovirus after 6-h exposure (reduction of 4.4 log units). No SODIS-induced reduction in A. polyphaga cyst viability was observed for sample temperatures below 45 degrees C. Total cyst inactivation was only observed after 6-h SODIS exposure at 50 degrees C (3.6 log unit reduction) and after 4 h at 55 degrees C (3.3 log unit reduction). CONCLUSIONS: SODIS is an effective means of disinfecting water contaminated with poliovirus and A. polyphaga cysts, provided water temperatures of 50-55 degrees C are attained in the latter case. SIGNIFICANCE AND IMPACT OF THE STUDY: This research presents the first SODIS inactivation curve for poliovirus and provides further evidence that batch SODIS provides effective protection against waterborne protozoan cysts.

Disinfection capacity of PuriLens contact lens cleaning unit against Acanthamoeba.

PURPOSE: The PuriLens contact lens system is indicated for cleaning and disinfection of soft (hydrophilic) contact lenses by means of subsonic agitation to remove lens deposits and microorganisms, and ultraviolet irradiation of the storage solution for disinfection. The capacity of the PuriLens system to disinfect storage solutions contaminated with known concentrations of Staphylococcus aureus, Pseudomonas aeruginosa, and Acanthamoeba species was evaluated. METHODS: An in vitro assessment of the antibacterial and antiparasitic efficacy of the PuriLens system was performed. Separated batches of the storage solution for the cleansing system were contaminated with stock strains of S. aureus and P. aeruginosa. A comparison of the microbiologic content was made between the solution before and after the cycle. RESULTS: The PuriLens system effectively eradicated S. aureus and P. aeruginosa organisms after a 15-minute cycle. However, viable cysts of acanthamoeba were recovered in the solution after the 15-minute cycle. CONCLUSIONS: The PuriLens system is highly efficient in protecting against contamination with common bacterial ocular pathogens. Acanthamoeba cysts, however, can survive in the solution or contact lens bath undergoing integrated subsonic debridement and indirect ultraviolet light disinfection. Use of chemical disinfecting solutions that contain agents such as chlorhexidine or other cationic antiseptics may be advisable in conjunction with use of the PuriLens device, especially in high-risk settings.

Phthalocyanine-photosensitized inactivation of a pathogenic protozoan, Acanthamoeba palestinensis.

Incubation of Acanthamoeba palestinensis cells with a tetracationic phthalocyanine (RLP068) at concentrations ranging between 0.2 and 1.0 microM, caused a ready uptake of the photosensitizer with recoveries of the order of 0.5-2.5 nmol per mg of cell protein. The amount of cell-bound phthalocyanine did not appreciably change with incubation times ranging between 0.5 and 3 h. Fluorescence microscopic investigations showed an obvious accumulation of the phthalocyanine at the level of the vacuolar membranes. A nearly complete photoinduced cell death occurred upon irradiating A. palestinensis cells with 600-700 nm light with a total energy of 15-30 J cm(-2) using 1.0 microM RLP068 in the incubation medium. DAPI staining of the photosensitized cells indicates significant damage of the nucleus. On the other hand, photosensitization of the protozoan cells does not directly involve the mitochondria as shown by the lack of photoinduced decrease in the activity of typical mitochondrial enzymes, such as NADH dehydrogenase and citrate synthase.

Resistance of Acanthamoeba castellanii cysts to physical, chemical, and radiological conditions.

Resistance of Acanthamoeba castellanii cysts to disinfection agents, antimicrobial agents, heat, freeze-thawing, ultraviolet radiation (UV), gamma irradiation, and cellulase were evaluated in vitro. Following exposure to different agents, the cysts were removed and cultured for A. castellanii trophozoites for 3-14 days. Solutions containing 20% isopropyl alcohol or 10% formalin effectively killed A. castellanii cysts. Hydrogen peroxide (3%, AOSept Disinfectant) effectively killed A. castellanii cysts after 4 hr of exposure. Polyhexamethylene biguanide (0.02%), clotrimazole (0.1%), or propamidine isethionate (Brolene) were effective in killing A. castellanii cysts in vitro. Acanthamoeba castellanii cysts were resistant to both 250 K rads of gamma irradiation and 800 mJ/cm2 of UV irradiation. Excystment of trophozoites was accelerated after exposure to 10, 100, and, 1,000 units of cellulase. These results suggest that A. castellanii cysts benefit by enhanced survival because of their resistance to very harsh environmental conditions.

Microwave treatment of contact lens cases contaminated with acanthamoeba.

PURPOSE: Microbially contaminated contact lens cases are a predisposing risk factor for Acanthamoeba keratitis. Several findings have shown that microwave irradiation kills the six Food and Drug Administration test challenge microorganisms. We aimed to determine what effect microwave irradiation has on Acanthamoeba trophozoites and cysts. METHODS: Different types of contact lens cases were contaminated with trophozoites and cysts of three different Acanthamoeba species (A. comandoni, A. castellanii, A. hatchetti) and were exposed to microwave irradiation for 3, 5, and 8 minutes, respectively. RESULTS: Trophozoites, as well as cysts of the different Acanthamoeba strains, were effectively killed, even by only 3 minutes of microwave irradiation, and there were no negative effects of irradiation on the contact lens cases themselves. CONCLUSION: We demonstrate that microwave treatment is a very effective, easy, and cheap method to keep contact lens cases free of Acanthamoeba, thus considerably reducing the risk of an Acanthamoeba keratitis.

Disinfection efficacy in an integrated ultraviolet light contact lens care system.

We evaluated a new integrated contact lens care system that combines fluid turbulence for lens cleaning with ultraviolet (UV) light for solution sterilization. The ultraviolet light system was used to clean and disinfect 42 soft contact lenses (water contents: 38.6%, 43%, 55%, and 70%) and two rigid gas permeable lenses. Test lenses were contaminated with 10(6) cells/mL of Bacillus pumilus, Aspergillus niger, Pseudomonas aeruginosa, and Acanthamoeba castellanii and subjected to a 15-minute cleaning-disinfection cycle. Bathing solutions and contact lenses were cultured at various time intervals and at the end of the cycle. All bathing solutions and all lenses but one were found to be sterile after one cycle. All units effectively disinfected solutions and contact lenses. This device may be an effective alternative to existing contact lens care systems.

Ultraviolet radiation for the sterilization of contact lenses.

Two sources of ultraviolet (UV) radiation with peak wavelengths in the UV-C or UV-B ranges were compared for their ability to sterilize contact lenses infected with Pseudomonas aeruginosa, Streptococcus pneumoniae, Acanthamoeba castellani, Candida albicans, and Aspergillus niger. Also examined was the effect of prolonged UV light exposure on soft and rigid gas permeable (RGP) contact lenses. The UV-C lamp (253.7 nm, 250 mW/cm2 at 1 cm) was germicidal for all organisms within 20 minutes but caused destruction of the soft lens polymers within 6 hours of cumulative exposure. UV-C caused damage to RGP lenses in less than 100 hours. The UV-B lamp (290-310 nm, 500 mW/cm2 at 1 cm) was germicidal for all organisms tested (except Aspergillus) with a 180-minute exposure and caused less severe changes in the soft lens polymers than did the UV-C lamp, although cumulative exposure of 300 hours did substantially weaken the soft lens material. RGP materials were minimally affected by exposure to 300 hours of UV-B. Ultraviolet light is an effective germicidal agent but is injurious to soft lens polymers; its possible utility in the sterilization of RGP lenses and lens cases deserves further study.

Contact lens disinfection by ultraviolet light.

A 253.7-nm ultraviolet light with an intensity of 1,100 microW/cm2 was tested for its germicidal activity against contact lenses and storage solutions contaminated with various corneal pathogens. The exposure time necessary to reduce a concentration of organisms from 10(6)/ml to less than 10/ml was 30 seconds for Staphylococcus aureus, 60 seconds for Pseudomonas aeruginosa, and 84 seconds for Candida albicans. The time necessary to sterilize a suspension of 10(4)/ml Acanthamoeba polyphaga was less than three minutes with this technique. Four brands of soft contact lenses were exposed to ultraviolet light for over eight hours without changing their appearance, comfort, or refraction.