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Adhesion of Acanthamoeba on Cosmetic Contact Lenses.

Background This study aimed to evaluate the adhesion of Acanthamoeba trophozoites on cosmetic contact lenses (CLs) with and without CL care multipurpose solution (MPS) treatment. Methods Acanthamoeba lugdunensis L3a trophozoites were inoculated onto disks trimmed from CLs: 1-day Acuvue moist, 1-day Acuvue define, Acuvue 2, and Acuvue 2 define. After 18-hour inoculation, the number of adherent trophozoites was counted under phase contrast microscopy. The effects of MPS, Opti-Free Express, soaking CLs for 6 hours, on Acanthamoeba adhesion were analyzed. Scanning electron microscopic examination was performed for assessment of Acanthamoeba attached on the lens surface. Results Acanthamoeba trophozoites showed greater adhesion to cosmetic CL (P = 0.017 for 1-day CL and P = 0.009 for 2-week CL) although there was no significant difference between the types of cosmetic CL. On all lenses, the number of adherent Acanthamoeba was significantly reduced after treatment with MPS (P < 0.001 for 1-day Acuvue moist, P = 0.046 for 1-day Acuvue define, P < 0.001 for Acuvue 2, and P = 0.015 for Acuvue 2 define), but there was still significant difference between conventional and cosmetic CLs (P = 0.003 for 1-day CL and P < 0.001 for 2-week CL, respectively). More attachment of Acanthamoeba was observed on colored area and the acanthopodia of Acanthamoeba was placed on the rough surface of colored area. Conclusion Acanthamoeba showed a greater affinity for cosmetic CL and mostly attached on colored area. Although MPS that contained myristamidopropyl dimethylamine reduced the adhesion rate, there was a significant difference between conventional and cosmetic CLs.


INTRODUCTION
Acanthamoeba keratitis occurs in contact lens (CL) users of all lens types, and can culminate in severe vision loss. The demand for cosmetic CLs continues to increase, particularly in teenage girls and young women all over the world, accounting for 13% of CL use in 2012, because they can be used to drastically alter the appearance of the eye. 1,2 For these reasons, the possibility of an increasing incidence of Acanthamoeba keratitis in cosmetic CL users should be considered. It has been reported that complications that can arise when wearing CL occur more frequently and are more serious in patients using cosmetic CLs than conventional CLs. 3 Adhesion of Acanthamoeba to CLs may play an important role in the pathogenesis of Acanthamoeba keratitis and depend on different parameters. Although many studies have reported the importance of hydrophobicity and surface roughness for microbial adhesion, analyses were mostly limited to noncolored conventional CLs. 4- 10 Recent studies have shown that the relative risk of developing CL related microbial keratitis is 16.5 times higher in cosmetic CL wearers than in conventional CL wearers, and suggested noncompliance as one of the most key factors. 3- 11 However, none of the studies have analyzed the characteristics of cosmetic CLs related to the adhesion of Acanthamoeba.
CL care multipurpose solutions (MPSs) for soft CLs comprise an aqueous liquid medium, an antimicrobial component, a surfactant, a buffer component, a viscosity-inducing component, a tonicity component, and a chelating component. 12 Although most CL wearers use MPSs because of their simplicity and convenience, several studies 13-18 conducted on the disinfecting efficacy of MPS against Acanthamoeba in conventional CLs showed that some MPSs had limited efficacies against Acanthamoeba trophozoites. It is thus important to determine the effects of MPSs on Acanthamoeba adhesion to cosmetic as well as conventional CLs.
Therefore, we investigated Acanthamoeba trophozoite adhesion in both cosmetic and conventional CLs and the efficacy of MPS disinfection against Acanthamoeba. We also used scanning electron microscopy (SEM) to examine adhesion patterns of Acanthamoeba trophozoites on the CL surface.

CLs
CLs were purchased from commercial sources and used for the adhesion assay. The properties of conventional (1-day Acuvue moist and Acuvue 2; Johnson & Johnson Vision Care, Inc., Jacksonville, FL, USA) and cosmetic (1-day Acuvue define and Acuvue 2 define; Johnson & Johnson Vision Care, Inc.) CLs are summarized in Table 1. All CLs were placed at the bottom of a 48-well plate (Falcon; BD Biosciences, Franklin Lakes, NJ, USA) with the convex side up.

Preparation of Acanthamoeba trophozoites
Although Acanthamoeba castellanii and Acanthamoeba polyphaga are common causative agents of Acanthamoeba keratitis in many countries, 19-21 this study examined Acanthamoeba lugdunensis, the most frequently isolated type of Acanthamoeba, from CL storage cases in Korea, 22-24 which were isolated from patients with Acanthamoeba keratitis and identified by riboprinting and 18S rDNA sequence analyses as previously described. Prior to an experiment, the medium was exchanged by rigorously shaking the culture bottles, removing the medium, and adding new medium. For the experiments, the number of Acanthamoeba applied to a substrate was determined by counting trophozoites beforehand using a Neubauer hemocytometer.

Adhesion assay of Acanthamoeba trophozoites to lenses
Lenses were placed in a 48-well plate filled with 1 mL of PYG. Acanthamoeba of 2 × 10 4 were added to each well containing a CL, followed by incubation for 18 hours. In the experiments, PYG medium was used to ensure that the Acanthamoeba was in a healthy state. After washing each well twice with 2 mL of phosphate buffered saline, the total number of trophozoites adhered to lenses was counted using a phase contrast microscope. Three lenses of each type were used in this experiment. Sixteen images per sample were taken on arbitrary positions and the number of trophozoites attached to each lens was counted.

Adhesion assay of MPS-treated Acanthamoeba to lenses
After soaking CLs for 6 hours with Opti-Free Express solution for testing, Acanthamoeba trophozoites of 2 × 10 4 in PYG were inoculated into a 48-well plate containing a lens. The total number of Acanthamoeba adhered to lenses was counted as described in the previous section. Three lenses were used for each type of lenses.

Statistical analysis
Statistical analysis of the number of adherent Acanthamoeba trophozoites to lenses was performed using analysis of variance with the Scheffe's test or non-paired t-test via SPSS (SPSS Inc., Chicago, IL, USA).

Adhesion of Acanthamoeba trophozoites to CLs with MPSs
After 18-hour inoculation, the number of adherent trophozoites on CLs was counted under phase contrast microscopy. We observed the adherence of trophozoites to cosmetic as well as conventional CLs and adhesion to cosmetic CLs was greater than that to conventional CLs ( Figs. 1 and 2). There was no significant difference between 1-day and 2-week CLs in either conventional or cosmetic CLs. Treatment with Opti-Free Express significantly reduced the number of adherent trophozoites in all CLs examined (P < 0.001 for 1-day Acuvue moist, P = 0.046 for 1-day Acuvue define, P = 0.0003 for Acuvue 2, and P = 0.015 for Acuvue 2 define), whereas there was still significant differences between conventional and cosmetic CLs even after Opti-Free Express treatment (Fig. 2).

Morphology of attached Acanthamoeba by SEM
SEM was performed for comparison of the lens surface of CLs. We found that the colored area was rougher than the uncolored area of cosmetic CLs (Fig. 3) and more Acanthamoeba trophozoites adhered to the surface of colored area (Fig. 4). The surface of cosmetic CLs to which amoebae adhere better is rough, which increases the contact surface with trophozoites, allowing firm attachment of acanthopodias ( Fig. 5A and B). Acanthamoeba treated with MPS attached firmly at the surface of cosmetic CL and was observed to be relatively round or a shrunken cystic shape, although the trophozoite of Acanthamoeba has a tendency of showing an elongated or slender shape (Fig. 5C).

DISCUSSION
Cosmetic CLs not only improve visual acuity but also change the appearance of the eye. Currently, many teenagers are exposed to cosmetic CLs and start to use them without proper education, and complications such as microbial keratitis occur more frequently and are more serious in patients using cosmetic CLs than in those using conventional CLs. 3 Acanthamoeba keratitis is a rare but potentially blinding infection associated with CL wear. CL use can have a negative impact on the eye's natural defenses and favor microbial colonization and survival. 28 A critical step before adhesion and invasion of the cornea is the adhesion to the CL. Although previous studies have demonstrated the ability of Acanthamoeba to stick to soft CL surfaces, little is known about their adherence properties to cosmetic CLs.
Numerous risk factors for CL related microbial keratitis have been described. Despite the progress in the quality of both CL materials 29,30 and sterile packaging, 29,31 the number of CL related microbial keratitis cases has remained roughly constant. 3, 32 The smoothness of and electrical charge on the CL surface have previously been shown to be related to bacterial adhesion. In particular, an increased surface roughness caused more bacterial adhesion. 9, 33 Bruinsma et al. 34 reported that the surface roughness was increased by CL wearing time, which was an important predictive factor for Pseudomonas aeruginosa adhesion to Etafilcon A lenses. Chan et al. 35 mentioned that surface pigments could increase the surface roughness of cosmetic CL and Ji et al. 36 found that significantly more Staphylococcus aureus and P. aeruginosa adhered to cosmetic CL than to conventional CL mainly on the colored surface rather than on the noncolored areas, which correlated with the surface roughness of CL. They concluded that bacterial adhesion and proliferation may be related to the increased microbial keratitis rate in cosmetic CL wearers because cosmetic CLs have more irregular and rougher surfaces than conventional CLs. In accordance with these studies, the adherence of Acanthamoeba trophozoites on cosmetic CL in this study was more than on conventional CL and might be related to the irregular surface of the cosmetic CL.
On SEM, the surface of cosmetic CL was more irregular and rougher compared with that of conventional CL. The mean lens surface roughness measured by atomic force microscopy was significantly higher for cosmetic CL than for conventional CL. 36 We confirmed that the noncolored surface of cosmetic CL was smoother and flatter than colored area, and the attachment of Acanthamoeba trophozoites to colored rough area was more than to noncolored smooth area. The attachment occurred through acanthopodia and a cystic pattern of Acanthamoeba trophozoites after MPS treatment was observed because Acanthamoeba changed its shape to survive under the poor condition, as we previously showed in the study of silicone hydrogel CLs. 37 Opti-Free Express, which was the most effective MPS in reducing attachment of Acanthamoeba on the silicone hydrogel CL surfaces, 37 was used in this study, and significantly reduced the numbers of adherent trophozoites in all lenses. However, the significant differences between conventional and cosmetic CLs even after Opti-Free Express treatment were still observed. In addition, there were also differences in the number of Acanthamoeba between 1-day and 2-week CLs with and without MPS treatment although they were not statistically significant. The colored area of 1-day lens was much rougher than that of 2-week lens and more Acanthamoeba trophozoites were observed in 1-day CLs than 2-week CLs. It may be argued that the adherence of bacteria to 1-day lenses is not of concern as they should be discarded after use. However, previous studies have reported that 1-day CLs did not reduce the risk of CL associated microbial keratitis. 30, 38 Hence, the importance of microbial adherence to daily disposable cosmetic CL should not be underestimated.
In summary, Acanthamoeba showed a greater affinity for the cosmetic CLs, interestingly on the colored surface, than on the conventional CLs, because cosmetic CLs have more irregular and rougher surfaces than conventional CLs, which could facilitate Acanthamoeba adhesion on the surface through acanthopodia. An MPS containing MAPD significantly reduced the adhesion rate on both conventional and cosmetic CLs, but there were still significant differences between these lenses even after MPS treatment. Awareness of increased propensity for Acanthamoeba adherence of cosmetic CLs would be useful for users as well as eye care professionals, and cosmetic CL wearers should be educated on the risks.