Comparative in vitro effectiveness of a novel contact lens multipurpose solution on Acanthamoeba castellanii

A. castellanii (ATCC® 50,370™) derived from human eye infection scrapings was acquired from ATCC (Manassas, VA). Trophozoites were propagated at 25 °C (room temperature) in yeast extract peptone dextrose (YPD) media augmented with 1% penicillin, streptomycin, and amphotericin B. Cultures were gently swirled for 96 h at 25 °C, and concentrations were quantified and verified using an automated coulter cell counter (Vi-Cell XR, Beckman Coulter Instruments, Indianapolis, IN) and diluted to 10⁴ cell/mL for use. In separate cocultures, cysts were induced via 72-h incubation of 100 μL 10⁶ cells/mL in 10 mL encystment media (95 mM NaCl, 5 mM KCl, 8 mM MgSO₄, 0.4 mM CaCl₂, 1 mM NaHCO₃, 20 mM Tris-HCl: pH = 9.0). Cysts were washed twice via centrifugation 5 min at 1000 rpm, enumerated using similar automated cell counting technique (Coulter), and then diluted with PBS to 10⁵ cells/mL for use.

Activity against the trophozoite form was evaluated via an Alamar Blue-based cellular respiration quantitative assay [22] and compared to currently marketed contact lens MPS Opti Free® (Alcon, Fort Worth, TX), BioTrue® (Bausch + Lomb, Bridgewater, NJ) or the newly developed solution ASP-57 (Asepticys, Alexandria, VA). Exactly 100 μL of media containing 10⁵ cells/mL Acanthamoeba castellanii cultures were commixed with 100 μL of either commercial or novel MPS solution with contact times ranging from 1 to 96 h before centrifugation for 10 min at 1000 rpm. The media-solution combinations were then aspirated, and 100 μL of fresh media added. Then, 10 μL of Alamar Blue (Reliablue™ Cell Viability Reagent (ATCC® 30-1014™, ATCC, Manassas, VA) was added and incubated. Absorbance was read after incubation at 570 nm via spectrophotometer. For statistical analysis of absorbance readings, unpaired T tests followed by F test to compare variances were performed using GraphPad Prism version 7.00 for Windows, GraphPad Software, La Jolla California USA.

Activity against Acanthamoeba cysts was determined via microscopic examination using Trypan blue to visualize cysts after treatment. Cysts were washed twice via centrifugation for 5 min at 1000 rpm, and diluted with PBS to 10⁵ cells/mL. Cysts and MPS formulations were each incubated at room temperature at a 1:1 ratio (10 μL:10 μL) using a duration of contact times (15, 30, 60, and 90 min). Thereafter, Trypan blue (10 μL) was used to visualize microscopically using a hemocytometer with a count of at least nine fields within the grid.

The bactericidal effectiveness of each solution was determined against Serratia marcescens (ATCC 13880) using assay methods described in ISO 14729 Stand-Alone Test criteria which requires that the solution must be capable of reducing bacterial viability by three logs (99.9%) within a particular timeframe. Briefly, S. marcescens was propagated on sheep’s blood agar (SBA) and were harvested using methods described in the ISO 14729 standard, and adjusted through centrifugation and dilution with PBS to a final concentration of 2.0 × 10⁵ CFU/mL. Exactly 100 μL of each microbial suspension (CFU) was added to 9.9 mL for each solution in polypropylene tubes under sterile condition. Mixtures were incubated at 25 °C for 60, 90, or 360 min. Thereafter, 1 mL of each mixture was diluted with a neutralizing broth (Gibco, Detroit, MI) and streaked onto SBA, where it was incubated at 32 °C for 48 h. Plates were enumerated using the spread plate method, and the log reductions calculated. All assays were performed in triplicate.

The multipurpose contact lens disinfection solutions BioTrue® (Bausch + Lomb, Bridgewater, NJ) and OptiFree® puremoist (Alcon, Fort Worth, TX) were acquired from commercial sources. The experimental MPS formulation, ASP-57, was obtained from Asepticys (Asepticys LLC, Alexandria, VA). The active ingredients of each solution tested are detailed in Table 1.

The alamar blue quantitative assay and protocol previously described [22] was initially confirmed and optimized within our laboratories prior to testing to ensure consistency within this particular test system, which included identification of the number of trophozoites to be used per well (3.25 × 10⁴ cells/well) to ensure proper reaction with the alamar blue reagent. In addition, the reciprocal of the absorbance values (570 nm) were used because the decrease of signal directly correlates to trophozoite viability and cellular respiration of the colorimetric reagent. Three contact times (1, 8, and 96 h) were used in separate assays. The results of the testing indicated (Fig. 1) that neither of the commercial solutions resulted in any substantial reduction in trophozoite viability at the 1 and 8 h timepoints, essentially matching that of PBS sham treatment. In contrast, the novel MPS ASP-57 resulted in significant reduction of trophozoites at the earliest and all other contact times attempted, indicating a complete kill subsequent to a 1 h contact time. Extending contact time to 8 or 96 h did not appreciably improve the efficacy of either commercial MPS; however, a significant decrease was observed in the 96 h Opti Free solution when compared to PBS sham treatment.

Trypan blue is commonly used as a vital stain to selectively color dead cells blue, whereas live cells with intact cell membranes are not colored. The dye exclusion method has worked effectively for viability with Acanthamoeba in past studies [23] and we utilized this method for direct microscopic counting of viable cysts on a hemocytometer for the current study. A number of contact times with the solutions under testing were attempted (15, 30, 60, and 90 min) and at least two individuals within the laboratory participated in counting. Collective results of the microscopic counts (Table 2) showed cysticidal activity of ASP-57 MPS at the earliest timepoint (15 min), effectively killing > 70% of cysts counted in comparison to PBS sham treatment. Killing effectiveness increased at later timepoints, with ASP-57 killing essentially all cysts (30 min, 96%; 60 min, 97%; 90 min 100%). In contrast, neither of the commercial solutions achieved a killing efficiency more than 35% at any of the timepoints attempted, with the exception of one (Opti Free, 51%) at 90 min contact time. Qualitative analysis of the effect of the different solutions on A. castellanii cysts (Fig. 2) clearly demonstrates the deleterious effects of exposure to the novel MPS ASP-57 (Fig. 2b) when compared to healthy, sham-treated (PBS) cysts (Fig. 2a). The exclusion dye (trypan blue) has been internalized in the cysts commixed with ASP-57, indicating that the plasma membrane has been completely compromised, with cellular contents proximal to the dead cyst (Fig. 2b). In contrast, cysts commixed with either of the commercial MPS show little change when compared to sham-treated samples (Fig. 2c, d), which is confirmed quantitatively in our enumeration (Table 2).

Serratia marcescens is one of five microorganisms included in the panel of agents required by ISO 14729 for testing the disinfection qualities of contact lens disinfection solutions. A three-log reduction capacity for the bacterial agents included in the panel at the minimum contact time of 4 h is considered consistent with acceptable disinfection capacity as ISO acceptance criteria. S. marcescens is considered the most resilient among the bacterial agents on the panel, and thus was chosen as an indicator organism for MPS potency control in the present study. The results of the testing (Fig. 3) indicate that the novel MPS ASP-57 achieved log reduction (> 5) at 60 min contact time compared to Biotrue (1.15) and Opti Free (1.33). ASP-57 produced a sterilizing effect (< 1 CFU; 5.30) at 90 min contact time; both commercial solutions failed to achieve minimum log reduction at this timepoint (Biotrue 2.36, Opti Free 1.84). Only after 6 h contact time did one of the two commercial solutions achieve the required 3-log reduction (Biotrue 3.70) while the other failed (Opti Free 2.10).