Evaluating Alternate Methods of Determining the Antimicrobial Efficacy of Contact Lens Care Products against Acanthamoeba Trophozoites

Abstract

Acanthamoeba keratitis (AK) is a serious ocular infection caused by a ubiquitous free-living amoeba, Acanthamoeba. This infection often results in extensive corneal damage and blindness, and is notoriously difficult to cure. While Acanthamoeba is an abundant organism, AK is most associated with contact lens hygiene noncompliance and inadequate contact lens care (CLC) disinfection regimens. Thus, accurate and timely antimicrobial efficacy testing of CLC solutions is paramount. Published methods for antimicrobial efficacy testing of Acanthamoeba trophozoites requires 14 days for results. Presently, alternate and/or rapid methods for evaluating CLC products rarely demonstrate equivalent results compared to commonly-reported methods. Propidium iodide is a cellular stain that can only bind to cells with damaged outer membranes. We evaluated propidium iodide staining as an alternative method for determining the relative antimicrobial efficacy of 11 different CLC products against Acanthamoeba trophozoites. Following exposure to a CLC product, the fluorescence intensity of propidium iodide in an Acanthamoeba population demonstrated a strong correlation to the log reduction determined by established, growth-based Acanthamoeba testing used to evaluate the antimicrobial efficacy of CLC products. Thus, propidium iodide was found to be an effective rapid tool for determining cell death in Acanthamoeba trophozoites following exposure to CLC solutions.

Differential Antimicrobial Efficacy of Multipurpose Solutions against Acanthamoeba Trophozoites

“…For three of these strains, representative images of the antimicrobial efficacy were obtained using a novel rapid method, which was recently outlined. 15 Propidium iodide, which stains red any cellular DNA components that are exposed because of cell death and loss of cell membrane integrity, or spilled because of cell lysis, was used to examine Acanthamoeba cultures immediately after completion of the multipurpose solution exposure time as a separate qualitative examination from the log reduction quantitative experiments. These representative examinations were performed for all seven multipurpose solutions with the ATCC 30461 strain (Fig.…”

“…As previously described, 14 , 15 axenic culture media (containing 20 g biosate peptone, 5 g glucose, 0.3 KH 2 PO 4 , 10 μg vitamin B 12 , and 15 mg l -methionine per liter of distilled deionized water) was used to produce homogenous populations of Acanthamoeba trophozoites. Axenic culture media was adjusted to a pH of 6.6 to 6.95 with 1 M of NaOH and autoclaved at 121°C for 20 minutes before storing at room temperature for use within 2 months.…”

“…To obtain images of the Acanthamoeba following disinfection efficacy of each solution, propidium iodide staining (as propidium iodide only binds to exposed cellular DNA of amoeba with damaged cell walls; Invitrogen, Carlsbad, CA) was used with three of the Acanthamoeba strains: ATCC 30461, ATCC 50370, and ATCC 50676. 15 Propidium iodide staining was performed on separate cultures from those used for quantification via the 50% endpoint method. Briefly, Acanthamoeba were seeded into a black clear-bottom 96-well plate at a density of 1 × 10 4 cells per well.…”

Continuous Real-Time Motility Analysis of Acanthamoeba Reveals Sustained Movement in Absence of Nutrients

“…As previously described [ 14 ], an axenic culture medium (AC6; containing 20 g biosate peptone, 5 g glucose, 0.3 g KH 2 PO 4 , 10 µg vitamin B12, and 1glass5 mg l -methionine per liter of distilled deionized water) was used to axenically produce Acanthamoeba trophozoites. AC6 was adjusted to a pH of 6.6–6.95 with 1 M NaOH and autoclaved at 121 °C for 20 min before being stored at room temperature for use within 3 months.…”

“…As the contact lens care community continues to improve amoebicidal disinfection efficacy and take steps to understand how Acanthamoeba may overcome ineffective contact lens care solutions, or how patient non-compliance with prescribed contact lens care regimens might lead to Acanthamoeba colonization of contact lens systems, many aspects of Acanthamoeba biology remain elusive. Therefore, this study seeks out to demonstrate the strength of live time-lapse Acanthamoeba imaging as a quantitative method to understand Acanthamoeba behavior and movements during the control conditions commonly used in ocular Acanthamoeba research [ 14 , 15 ]. The control conditions examined here are combinations of commonly-used surfaces and solutions: glass with Ringer’s solution, glass with E. coli , glass with an axenic culture medium, and non-nutrient agar with Ringer’s solution.…”