Flow cytometry for determination of the efficacy of contact lens disinfecting solutions against Acanthamoeba spp.
Flow cytometric analyses of cellular staining with fluorescent viability dyes and direct microscopic observations of methylene blue exclusion were compared for evaluation of the effects of a chlorhexidine gluconate-based contact lens disinfectant solution and a polyhexamethylene biguanide solution against cysts and trophozoites of Acanthamoeba castellanii and Acanthamoeba polyphaga. The flow cytometric procedure with propidium iodide (used to stain dead cells) indicated that more than 90% of trophozoites of both species (inocula of 10(5) to 10(6)/ml) at 22 degrees C lost their viability after 4 h of exposure to chlorhexidine. When propidium iodide was used in combination with fluorescein diacetate (for live cells), the apparent number of propidium iodide-stained cells was reduced, but the relative efficacies of the two biguanide solutions appeared unchanged from those evident with the single dyes; the chlorhexidine solution was more effective than the polyhexamethylene biguanide solution. Similar data were obtained with the more cumbersome methylene blue exclusion procedure. Flow cytometric analyses provided a statistically reproducible and rapid procedure for determining the relative antiamoebal efficacies of the disinfecting solutions. (+info)
Efficacy of a contact lens cleaning device and its enhancement of the performance of contact lens care products.
BACKGROUND: Corneal infections due to contact lens contamination are risks associated with contact lens wear. Care systems which reduce these risks are desirable. METHODS: This study evaluated a contact lens cleaning device using normal saline initially and then four contact lens solutions. RESULTS: Using saline, six out of 10 tests resulted in complete removal of challenge organisms or showed reductions to 10 cfu/ml or <10 cfu/ml. Tests upon organism using multipurpose solutions showed >6 log reductions in 2-3 minutes. With laboratory made biofilms, similar results were obtained. CONCLUSIONS: This device improves lens cleaning and enhances lens care solutions. When used correctly it should lead to significant reductions in microbial keratitis associated with inadequate contact lens hygiene. (+info)
A comparison of cyst age and assay method of the efficacy of contact lens disinfectants against Acanthamoeba.
AIMS: (i) To determine effect of Acanthamoeba cyst age, method of production, and (ii) to assay technique on the efficacy of multipurpose solutions (MPS) and hydrogen peroxide based contact lens disinfectants. (iii) To establish if MPS can remove mature cysts from contact lenses according to the ISO/DIS 14729 regimen test for microbe removal. METHODS: Immature and mature cysts of A polyphaga were tested against the MPS Opti-Free express and the hydrogen peroxide based solutions Oxysept 1Step and Oxysept 1 using two assay methods. Simulated patient regimen testing was performed with the Opti-Free express and Complete using mature cysts inoculated on to group I or group IV lenses. RESULTS: Immature cysts were sensitive to disinfection by all solutions. No killing was observed with mature cysts with Opti-Free express, while immature cysts yielded a 1-2 log reduction in viability. Oxysept 1Step gave a 1.1 (SD 0.3) log reduction in mature cysts after 6 hours. Oxysept 1 gave a 2.4 (0.3) log reduction in mature cysts after 4 hours and a 3.8 (0.5) log reduction after 6 hours. Patient regimen testing using Opti-Free express and Complete resulted in no recovery of viable mature cysts from the contact lenses or from the soaking solutions. CONCLUSION: Cyst age but not method of production used in this study influences the efficacy of contact lens disinfectants against Acanthamoeba. MPS are effective in removing cysts from contact lens surfaces and may have a role in the prevention of acanthamoeba keratitis. (+info)
Resistance of Pseudomonas aeruginosa isolates to hydrogel contact lens disinfection correlates with cytotoxic activity.
One of the most common pathogens in infection of hydrogel contact lens wearers is Pseudomonas aeruginosa, which can gain access to the eye via contamination of the lens, lens case, and lens care solutions. Only one strain per species is used in current regulatory testing for the marketing of chemical contact lens disinfectants. The aim of this study was to determine whether P. aeruginosa strains vary in their susceptibility to hydrogel contact lens disinfectants. A method for rapidly screening bacterial susceptibility to contact lens disinfectants was developed, based on measurement of the MIC. The susceptibility of 35 P. aeruginosa isolates to two chemical disinfectants was found to vary among strains. MICs ranged from 6.25 to 100% for both disinfectants at 37 degrees C, and a number of strains were not inhibited by a 100% disinfectant concentration in the lens case environment at room temperature (22 degrees C). Resistance to disinfection appeared to be an inherent rather than acquired trait, since some resistant strains had been isolated prior to the introduction of the disinfectants and some susceptible P. aeruginosa strains could not be made more resistant by repeated disinfectant exposure. A number of P. aeruginosa strains which were comparatively more resistant to short-term disinfectant exposure also demonstrated the ability to grow to levels above the initial inoculum in one chemical disinfectant after long-term (24 to 48 h) disinfectant exposure. Resistance was correlated with acute cytotoxic activity toward corneal epithelial cells and with exsA, which encodes a protein that regulates cytotoxicity via a complex type III secretion system. These results suggest that chemical disinfection solutions may select for contamination with cytotoxic strains. Further investigation of the mechanisms and factors responsible for resistance may also lead to strategies for reducing adverse responses to contact lens wear. (+info)
Viability of Acanthamoeba after exposure to a multipurpose disinfecting contact lens solution and two hydrogen peroxide systems.
BACKGROUND/AIM: Contact lens cases contaminated with Acanthamoeba are a major risk factor for an infection of the eye. In this study the anti-Acanthamoeba activity of three different contact lens storage solutions was tested. METHODS: A new multipurpose contact lens storage solution (Meni Care Plus) and a two step (Titmus H(2)O(2)) and one step (Oxysept Comfort) hydrogen peroxide system were tested for their effects on trophozoites and cysts of three different Acanthamoeba species: A castellanii, A hatchetti, and A lenticulata. RESULTS: After a soaking time of 8 hours (overnight soaking of contact lenses) the Titmus H(2)O(2) 0.6% solution showed very good amoebicidal effects, while Oxysept Comfort 3% H(2)O(2) could not effectively destroy the cysts of any of the three tested species. Viable cysts of the species A lenticulata and A hatchetti were still present after exposure to Meni Care Plus (0.0005% PHMB) for 8 hours. CONCLUSION: Not all of the three tested contact lens storage solutions have sufficient amoebicidal effects. The two step peroxide system Titmus H(2)O(2) is a very effective disinfectant contact lens solution in order to avoid a possible Acanthamoeba infection of the eye. (+info)
Phagocytosis affects biguanide sensitivity of Acanthamoeba spp.
The incidence of Acanthamoeba keratitis, a disease associated with contact lens wear, has been in apparent decline with the advent of multipurpose contact lens solutions. The concentrations of the biguanides chlorhexidine digluconate (CHX) and particularly polyhexamethylene biguanide (PHMB) included in multipurpose solutions (MPSs) are sublethal for amoebae. We evaluated by flow cytometry the effects of these two biguanides on phagocytosis of particles and the survival of trophozoites of Acanthamoeba castellanii and A. polyphaga. Trophozoites of A. castellanii and A. polyphaga (10(6)/ml) were exposed to solutions of 5 and 50 microg of PHMB and CHX per ml in the presence and absence of particles (i.e., heat-killed yeasts and bacteria and latex beads). In addition, trophozoites were exposed to particles treated with these concentrations of the two biguanides. In the absence of particles, trophozoites of A. polyphaga appeared to be more resistant to the biguanides than those of A. castellanii. In the presence of particles, the rates of survival of both species were decreased. In most instances, particles treated with sublethal concentrations of both biguanides that were adsorbed onto the particles reduced the incidence of phagocytosis. Particles present in MPSs in contact lens cases may be involved in the decreased incidence of Acanthamoeba keratitis. (+info)
Aspects of the antimicrobial mechanisms of action of a polyquaternium and an amidoamine.
OBJECTIVES: Polyquad (Alcon) (polyquaternium-1, PQ-1) and Aldox (Alcon) (myristamidopropyl dimethylamine, MAPD) are two biocides that are used commercially in a contact lens disinfecting solution, namely Opti-Free Express (Alcon) multi-purpose disinfecting solution. Their potential mechanisms of action were investigated against a range of common ocular pathogens. These were Acanthamoeba castellanii (trophozoites and cysts), Aspergillus fumigatus, Candida albicans, Pseudomonas aeruginosa, Serratia marcescens and Staphylococcus aureus. METHODS: Three aspects were investigated: the lethal effects of the biocides on the organisms, the leakage of K+ from treated cells, and the lysis of spheroplasts derived from the cells. RESULTS: PQ-1 was found to have predominantly antibacterial activity, and induced K+ leakage from the bacteria and C. albicans. It also caused lysis of spheroplasts of S. marcescens, but not those of C. albicans. MAPD was active against all of the organisms, but showed higher activity against the fungi and amoeba. It induced K+ leakage from A. fumigatus and C. albicans, and like PQ-1, lysed the spheroplasts of S. marcescens but not C. albicans. CONCLUSIONS: The two biocides have different spectra of antimicrobial activity. PQ-1 has mainly antibacterial activity, whereas MAPD was active against all of the test organisms, particularly the fungi. (+info)
Determination of amoebicidal activities of multipurpose contact lens solutions by using a most probable number enumeration technique.
Six multipurpose contact lens solutions [All-in-One, All-in-One (Light), ReNu MultiPlus, Optifree Express, Complete, and Solo-care soft] were tested for their efficacies against Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique for amoebic enumeration. Against trophozoites, All-in-One, ReNu Multiplus, and Optifree Express achieved total kill (log reduction of >3) after the manufacturer's minimum recommended disinfection time (MMRDT), with the remaining solutions failing to reach a log reduction of 1. After 24 h of exposure, all solutions proved trophozoiticidal, achieving, with the exception of Complete (log reduction of 3.13), total kill. Against cysts, All-in-One gave a log reduction of >3 within the MMRDT, with all other solutions failing to achieve a log reduction of 1. After 24 h of exposure, All-in-One achieved total kill of cysts (log reduction of 3.74), ReNu MultiPlus gave a log reduction of 3.15, and the remaining solutions reached log reductions of between 1.09 and 2.27. The MPN technique provides a simple, reliable, and reproducible method of amoebic enumeration that depends on simply establishing the presence or absence of growth on culture plates inoculated with a series of dilutions and determining the MPN of amoebae present from statistical tables. By use of this technique, two of the multipurpose solutions tested, ReNu MultiPlus and Optifree Express, demonstrated effective trophozoiticidal activities within the recommended disinfection times; however, only All-in-One proved effective against both trophozoites and cysts over the same time period. This MPN technique, which uses axenically produced trophozoites and mature, double-walled cysts, has the potential to form the basis of a national standard for amoebicidal efficacy testing of multipurpose contact lens disinfecting solutions. (+info)