Contact lenses worn more than once are usually subjected to a regimen of cleaning and disinfection between periods of wear. Multipurpose disinfection solutions (MPDSs) are currently the most commonly used care regimens to clean and disinfect soft contact lenses, representing ∼90% of all lens care systems prescribed.1,2 MPDSs are designed to clean, disinfect, and rewet the lens surface in a single process. The majority of the MPDSs were initially introduced for use with conventional soft contact lenses by using manual rub and rinse steps before overnight soaking of the lenses. Early studies demonstrated that rub and rinse steps in a regimen were essential to improve the efficacy of contact lens disinfectants.3 Digital rubbing of the lens also removes lens deposits, which may result in discomfort and facilitate accumulation of microorganisms on the lens surface. Rub and rinse effectively removes loosely bound deposits,4,5 although protein removal efficiency depends on the lens material and protein type.6
To satisfy consumer's wishes for reduced time and complexity of the lens care procedures and improved convenience, the trend for contact lens care products has been toward fewer and shorter regimen steps. No-rub MPDSs have been developed and dominate the contact lens care market. However, microbes are often more resistant in biofilm formed on surfaces.7 Furthermore, with the increasing use of silicone hydrogel lenses,8 it is unclear how MPDSs perform once microbes have adhered to these lens types. Studies comparing the effect of rub and no-rub on disinfection efficacy during silicone hydrogel lens care are scarce.9 The aim of the study was to evaluate the effect of lens care regimen steps on the efficacy of MPDSs when used with silicone hydrogel and conventional lenses.
MATERIALS AND METHODS
Microorganisms and Culture Preparation
The panel of microorganisms recommended in the ISO 14729 standard10 such as Staphylococcus aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Serratia marcescens ATCC 13880, Candida albicans ATCC 10231, and Fusarium solani ATCC 36031 were used in the study. Acanthamoeba polyphaga Ros was also included in the study.
Challenge bacterial strains were grown on Trypticase Soy Agar (TSA, soybean-casein digested agar) at 37°C for 18 to 24 h. Yeast strains were grown on Sabouraud Dextrose Agar at 37°C for 24 h and molds on Potato Dextrose Agar (PDA) at 25°C for 7 to 10 d. Acanthamoeba strain was grown and maintained in Peptone Yeast Extract Glucose medium at 30°C for 3 to 5 d to generate trophozoites.
For preparation of inocula, bacteria, yeast, and mold cells were harvested and resuspended in sterile phosphate buffered saline containing 0.05% tween 80 (PBST; pH 7.3) and adjusted to the final concentration of 1.0 × 108 colony forming units per mL (CFU/mL; OD of 0.1 at 660 nm). Acanthamoeba trophozoites were harvested, resuspended in Page saline and adjusted to a final concentration of 107 cells/mL using a hemocytometer.
Contact Lens and Lens Care Products
Commercially available silicone hydrogel and soft hydrophilic contact lenses were used throughout the study including O2OPTIX (lotrafilcon B, FDA group I, CIBA Vision), ACUVUE ADVANCE (galyfilcon A, FDA group I, Johnson & Johnson), and ACUVUE 2 (etafilcon A, FDA group IV, Johnson & Johnson). The three commercially available contact lens MPDSs used in the study were OPTI-FREE Replenish [Alcon, containing polyquaterium-1 (PQ-1) & myristamidopropyl dimethylamine (MAPD)], AQuify [CIBA Vision, containing polyhexamethylene biguanide (PHMB)], and ReNu MultiPlus (Bausch & Lomb, containing PHMB). The product codes and a brief description of the products are shown in Table 1.
Contact Lens Inoculation and Disinfection Regimens
Each lens was inoculated with a challenge microorganism prepared in PBST (∼1 × 108 CFU/mL for bacteria and fungi, and 1.0 × 107 cells/mL for Acanthamoeba) in a volume of 20 μL per lens (10 μL per side). The organisms were allowed to adhere to the lens for 10 min; then one of the regimens was implemented, including “rub and rinse,” “rinse-only,” and “no rub and no rinse.” The treated lens was then placed into a well of a 12-well tissue culture plate containing 3 mL (consistent with the lens case volume) of lens care product. The lenses were then soaked for 4 or 6 h based on labeled minimum disinfection time for each solution.
Enumeration of Viable Organisms
After disinfection, bacterial and fungal survivors on lens surfaces were enumerated using 10-fold serial dilution and agar overlay methods. Each lens was transferred to a 5 mL plastic vial containing 2 mL of Dey/Engley Neutralizing Broth (DENB) and a small magnetic stirring bar. The lens was vortexed at maximum speed for 60 s to detach adherent cells. The lens homogenate was 10-fold serially diluted in DENB and plated out onto TSA plates containing neutralizers (TSA-N, 0.07% lecithin and 0.5% polysorbate 80) for bacterial recovery and onto PDA for fungal recovery after appropriate incubation periods. Each lens was plated separately in an agar overlay of TSA-N or PDA. For Acanthamoeba recovery, 10-fold serially diluted lens homogenates were inoculated in quadruplicate into wells of six-well tissue culture plates containing non-nutrient agar seeded with E. coli. Plates were examined under the microscope for the presence of replicating cells after 3 and 7 d of incubation at 30°C. Positive or negative growth on each well was recorded at each examination point. Final Acanthamoeba counts from each test product were determined using the Reed-Muench computation table.11
To enumerate viable organisms in the soaking solutions, serial dilution and membrane filtration methods were used. Soaking solutions were transferred to a vial containing 25 mL PBST. The mixtures were filtered with a 0.22 μm membrane (GV, Durapore Membrane Filters, Millipore) and plated on an appropriate agar surface. The soaking solution was also 10-fold serially diluted in DENB and plated on appropriate agar plates if necessary for recovery of bacteria and fungi. For enumerating remaining Acanthamoeba in soaking solution, the serially diluted solutions were plated out and the final counts were determined using the method described above.
The total survivors per lens for each organism were calculated by summing numbers of organisms recovered from the lens surface and the soaking solution. All the regimen tests were conducted in laboratory settings and were randomized but unmasked.
Univariate analysis of variance was performed with log transformed CFUs or cell numbers to assess the difference in numbers of remaining organism following use of the lens care products and the interaction between the test regimens, products, and contact lens types. Post hoc multiple comparison using Tukey correction was performed to assess the differences within regimens, products, or lens types. Statistical significance was set at p < 0.05.
Effectiveness of Regimen Procedures on Disinfection
As shown in Fig. 1, lenses treated by soaking only, correlating to the “no rub and no rinse” regimen, demonstrated the highest numbers of microbial survivors compared with the other treatment regimens (p < 0.0001). Only 34% of lenses exposed to bacteria, 4% of lenses exposed to Acanthamoeba, and none of the lenses exposed to fungi had no more than 10 survivors. “Rinse-only” plus soaking significantly reduced the numbers of microorganisms on lens surfaces compared with that on lenses treated with “no rub and no rinse” regimen (p < 0.0001). After rinsing and soaking, 67% lenses challenged with bacteria, 38% lenses with fungi, and 66% lenses with Acanthamoeba showed ≤10 CFU organisms per lens. The lowest overall numbers of microbial survivors were found on contact lenses treated with a “rub and rinse” step before soaking in MPDS compared with other treatment regimens regardless of MPDS types and lens types (p < 0.0001). When a full regimen of “rub and rinse” plus soaking was applied, 99% of lenses challenged with bacteria, 75% with fungi, and 89% with Acanthamoeba showed ≤10 CFU per lens.
Performance of Multipurpose Solutions with Regimen Procedures
Table 2 details the number of surviving organisms on lens surfaces after treatment with various multipurpose solutions under regimens. In “no rub and no rinse” and the “rinse-only” regimens, solutions containing PQ-MAPD generally performed better than other products against overall challenge microorganisms (p < 0.0001, regardless of microorganism type). There was no difference in the antimicrobial efficacy of the two solutions containing PHMB (PHMB-1 and PHMB-2; p = 0.874). Under the “no rub and no rinse” regimen, 30% of lenses treated with PQ-MAPD and 11% of lenses treated with PHMB showed ≤10 CFUs (p < 0.001). However, when examining differences between solutions for each of the different microorganisms, the numbers of fungi remaining on the lenses soaked in the PQ-MAPD without rubbing and rinsing (“no rub and no rinse”) were not significantly different to that on the lenses soaked in either PHMB solution (p = 0.353). In the “rinse-only” regimen, 81% of lenses treated with PQ-MAPD vs. 49% of lenses treated with either PHMB solution (p < 0.001) passed the regimen test criterion (≤10 CFUs). When “rub and rinse” steps were applied, there were no differences in the antimicrobial efficacy between solutions (p = 0.275). Similarly, the percentages of the lenses with ≤10 CFUs were not significantly different between the groups treated with various MPDSs, ranging from 87 to 93%.
Effect of Contact Lens Materials on the Efficacy of Disinfection
Table 3 displays average microbial survivors per lens on each lens type following treatment with different regimens regardless of MPDS types. There were significantly fewer bacteria, remaining on galyfilcon A than on other lens surfaces when treated with the “no rub and no rinse” or the “rinse-only” regimens (p = 0.027). Thirty-two percent of galyfilcon A vs. 8% of lotrafilcon B and 13% of etafilcon A lenses had CFUs ≤10 (p < 0.0001) when lenses treated with the “no rub and rinse” regimen; whereas under the same regimen, the average number of fungal and Acanthamoeba survivors were not significantly different among all types of lenses (p = 0.671). When lenses were rinsed and soaked, the average number of microorganisms including fungal and Acanthamoeba survivors were lower on galyfilcon A (1.0 log CFUs) than on lotrafilcon B (1.8 log CFUs, p < 0.0001) or etafilcon A lenses (1.4 log CFUs, p = 0.007). Eighty-one percent of galyfilcon A lenses showed ≤10 CFUs compared with 44% and 55% of lotrafilcon B and etafilcon A lenses, respectively, with ≤10 CFUs (p < 0.0001). The regimen of “rub and rinse” followed by soaking of lenses resulted in almost complete removal/killing of challenge bacterial strains (average of 0 to 0.1 log CFU per lens) on various lens type, and there were no significant differences in the numbers of surviving organisms between these lens types (p = 0.064).
Appropriate care of contact lenses and lens cases may limit microbial contamination. Use of contact lens care products following the labeling instructions of the manufacturers may provide increased assurance that viable populations of contaminating microorganisms will be reduced. Label instructions are usually designed to direct the wearer in appropriate care for their lenses, including regimen steps of disinfection and storage times. Before marketing, contact lens disinfectants are required to be evaluated by a Stand-Alone test procedure for antimicrobial efficacy against representative reference organisms defined by the International Organization for Standardization (ISO) standard.10 There is also a regimen test in the ISO standard for evaluation of products and regimens for hygienic management of contact lenses.10 The criteria of the regimen test are that solutions must result in ≤10 CFU survivors per lens following the regimen procedure recommended by manufacturers for lens care. However, the regimen test is not required if the product passes the primary Stand-Alone test criteria.
This study has demonstrated that a “rub and rinse” step before disinfection is the most effective regimen with every MPDS to reduce the number of surviving microbes on contact lenses. When the full “rub and rinse” and disinfection regimen was used, the average number of surviving microorganisms per lens was ≤10 CFU for all the lenses and solutions tested. These results are generally in agreement with the previous findings, where “rub and rinse” regimes, with or without a disinfection step were effective in removing/killing large numbers of bacteria, fungi, and Acanthamoeba from conventional contact lenses12–15 or silicone hydrogel lenses,9 and met the ISO 14729 regimen test criteria. When the rubbing step was omitted, as may be recommended by manufacturers, the efficacy, particularly antifungal activity of the test products, was decreased, and none of the solutions met the regimen test criteria. It has been shown previously that rinsing before disinfection of soft hydrophilic contact lenses is important to the disinfection efficacy of PQ-MAPD-containing solutions and most of the PHMB-containing solutions, resulting in a few to no survivors.15 Our findings generally support the assertion that use of rinsing and soaking, without rubbing, is not effective in removing pathogenic microbes from silicone hydrogel lenses.9,12–16 When lenses were disinfected with the “no rub and no rinse” regimen, as recommended for certain MPDS (e.g., the PHMB-1-containing solution), none of the test solutions passed the regimen test criteria. Similar findings have been reported by other investigators.9,15
Disinfection efficacy of the MPDS with the regimens was also dependent on the type of lens material used. The observation that bacteria adhered to galyfilcon A lenses were more susceptible than other lens types to the disinfection of MPDSs under various regimens, suggests that bacteria may be more loosely bound to the galyfilcon A lens. Thus, when rinsed or soaked in MPDS, the organisms can be easily removed or detached and become susceptible to disinfectant. The magnitude of adhesion of microorganisms to contact lenses differs according to the types of lens materials.17–19 Adhesion of bacteria to unworn conventional hydrogel lenses (Etafilcon A) has been shown to be significantly lower than that to silicone hydrogel contact lenses.17,20 Additionally, bacterial removal has been found to be more likely from the surface of galyfilcon A and etafilcon A lenses than from lotrafilcon A or B lenses when exposed to PHMB-2 in vitro.18 Galyfilcon A and lotrafilcon A and B are silicone hydrogel lenses. The apparent lack of firm adhesion of microorganisms to the galyfilcon A compared with the lotrafilcon B lens may be due to the polyvinylpyrrolidone used as an internal wetting agent in galyfilcon A, while a surface coating with plasma is applied to lotrafilcon B lens. Although our results revealed that certain lens materials were more difficult to disinfect once contaminated with bacteria in vitro, as soon as a contact lens is placed in the eye it becomes rapidly conditioned by the tear film and this may also influence bacterial colonization.21–23 Worn conventional hydrogel lenses are more prone to bacterial adhesion than worn silicone hydrogel materials.24 In this context, rubbing and rinsing the contact lens after removal from the eye also becomes important in effectively removing/cleaning tear deposits from the worn lens.4,5
This study demonstrated that a yeast, mold, and Acanthamoeba were less susceptible to the rinsing/cleaning and disinfection by MPDSs compared with bacteria once they attached to lens surfaces. Biofilms of Candida and Fusarium are tightly bound to hydrogel and silicone hydrogel contact lenses.25 Fusarium isolates may penetrate both conventional and silicone hydrogel lenses,26–28 and clinical isolates of Fusarium possess a greater capacity than a standard test strain to attach to and penetrate lenses, and to survive exposure to various MPDS.26–29 Even in the stand-alone test for measuring MPDS disinfection in the absence of a contact lens, clinical and environmental isolates are often less susceptible to MPDSs compared with laboratory strains.25,29–34 Acanthamoeba exhibit a significantly greater affinity for lotrafilcon A35,36 and B,37 or balafilcon A19 lenses compared with conventional hydrogels or galafilcon A lenses.35 Using a no-rub/rinse regimen test, most of MPDSs have been shown to remove/kill Acanthamoeba adhered to conventional hydrogel lenses but not from certain silicone hydrogel lenses.38 Interestingly, one report suggests that the only MPDS that passed the criteria for no-rub/rinse regimen tests when used with silicone hydrogel lenses was ReNu with MoistureLoc,38,39 which has now been removed from the market because of problems with activity against Fusarium sp.40,41 Recent outbreaks of microbial keratitis in contact lens wearers have been attributed to inadequate MPDS efficacy against certain pathogens including Fusarium and Acanthamoeba, and inappropriate lens care hygiene of lens wearers.40–46 Nevertheless, similar to previous reports, our study strongly suggest that eliminating the digital rub may compromise the cleaning and disinfecting efficacy of the MPDS during use and eliminate a critical preventative technique in managing microbial keratitis.
In conclusion, the current studies have demonstrated that antimicrobial efficacy of the contact lens MPDS is dependent on lens care regimens. Only the full regimen of rubbing and rinsing before disinfection was effective for all MPDSs. We believe this step will help ensure lens disinfection against all types of microorganisms, and subsequently prevent colonization of the eye by pathogenic organisms, thus providing protection to contact lens wearers. “Rub and rinse,” in conjunction with soaking of lens, is the most effective regimen to recommend for all the multipurpose lens care solutions used with any type of contact lenses.
We thank Dr. Judith Flanagan, the Brien Holden Vision Institute, Sydney, Australia, for assistance with the preparation of the manuscript.
Brien Holden Vision Institute
Level 5, North Wing
Rupert Myers Building
University of New South Wales
Sydney, NSW 2052
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