The past 10 years has seen a dramatic shift in the types of contact lenses and contact lens disinfecting solutions available. Silicone hydrogel lenses were first released around the world in the late 1990s, early 2000s. Since that time, there has been an increase in almost all markets of the use of these lenses, and they now make up the majority of new lens fits, with lenses being used on a daily or extended/continuous wear basis.1 During daily wear, all lenses need to be disinfected and cleaned when not in the eye. The most widely used contact lens disinfecting and cleaning solutions contain a variety of disinfectants including polyquaternium-1, myristamidopropyl dimethylamine (Aldox), polyhexamethylene biguanide (also known as polyhexanide), and hydrogen peroxide and various cleaning agents including surfactants. The rate of use of multipurpose disinfecting solutions has gradually increased to account for >90% of disinfection types in United Kingdom and Australia.2,3
In epidemiological studies of contact lens wear over the past 20 years, even with the release of new disinfecting solutions and new lens types/materials, there has been an almost constant rate of microbial keratitis associated with wear. The initial estimates, published in 1989, for the rates of microbial keratitis during daily wear and for extended wear are almost identical to those published in 1999 and 2008.4–6 The risk factors associated with microbial keratitis include overnight use, poor storage case hygiene, smoking, Internet purchase of contact lenses, <6-month wear experience, and socioeconomic class.6 For daily-wear lenses only, the risk factors that have been proposed include male gender,4,7,8 smoking,8 no or infrequent lens disinfection,7,9,10 use of chlorine- or heat-based disinfection systems,9 having diabetes,11 no surfactant in the disinfecting solution or no use of rub/rinse step during lens cleaning/disinfection,11 noncompliance with the hygiene regime,12 and reduction in case cleaning.13 Poor storage case hygiene has an odds ratio of 3.70 compared with good case hygiene from an Australian study.6 However, in a study conducted in the United Kingdom, hygiene (including lens case hygiene) was not a significant factor.14 On balance, there is good evidence that the contact lens storage case is often a significant factor associated with microbial keratitis. Furthermore, microbial contamination of contact lens storage cases is also associated with sterile infiltrates in the cornea.15
Microbial contamination of contact lens storage cases has been estimated and can range from 53 to 57%16–18 to 81 to 83%.19,20 In a smaller scale 1-month trial, Seal et al.21 found that only 22% of cases were contaminated when using a multipurpose solution. Gray et al.20 found that contamination of cases with bacteria was common (77%), followed by contamination with fungi (24%) and then protozoa (20%). Stapleton et al.22 found that bacterial contamination of cases was more likely to occur with wearers who were suffering from microbial keratitis (85%) vs. wearers with no keratitis (36%). Dutot et al.23 demonstrated that multipurpose disinfecting solutions were used by ∼59% of a sampled population from France and 35% used “oxidative products” (presumably mostly hydrogen peroxide disinfecting solutions), whereas 80% of people with severe corneal infection had used multipurpose solutions, suggesting an over-representation of these latter solutions in people with microbial keratitis.
The aim of the current investigation was to assess the contamination rate and level in contact lens cases of wearers for three commercially available silicone hydrogel lenses and users of four different disinfecting solutions. Effects of the different lenses or solutions on the rate and level of contamination were evaluated.
MATERIALS AND METHODS
Participants, Contact Lenses, and Solutions
Contact lens cases were collected from a series of ongoing, nonrandomized, dispensing, clinical studies conducted at the Institute for Eye Research, Sydney, Australia. Lenses made from senofilcon A (ACUVUE OASYS; Johnson and Johnson Vision Care, FL), comfilcon A (Biofinity; Coopervision, NY), or balafilcon A (PureVision; Bausch and Lomb, NY) were worn on a daily basis for 3 months by 232 participants. Lenses were disinfected using one of four care systems whose formulations are listed in Table 1. Approximately 40 participants were enrolled for each lens/solution combination (OASYS/ClearCare; PureVision/AQuify; Biofinity/ClearCare; Biofinity/AQuify; Biofinity/Opti-Free Express; and Biofinity/ Opti-Free RepleniSH). All procedures were conducted in accordance with the tenets of the Declaration of Helsinki 2000 and approved by the Local Area Health Ethics Committee. Participants were advised of any potential adverse reactions and signed a record of informed consent before enrolment.
Participants were advised to wear the lenses on the manufacturers recommended schedule (monthly replacement for Biofinity and PureVision; every 2 weeks for OASYS). For solutions, participants were advised to rinse lenses for 5 s each side with the solution (or unit-dose saline in the case of ClearCare) before storing in solution overnight and to place lenses on the eye straight from the case the next morning. Users of ClearCare (CIBA Vision, GA) were given the lens case appropriate for that solution as supplied by the manufacturer (i.e., vertical bottle containing basket to hold lenses and inbuilt platinum disk for neutralization). Users of all other solutions were given a flat polypropylene case (as manufactured by CIBA Vision).24 AQuify solution is supplied by the manufacturer (CIBA Vision) with a lens case that contains silver. We chose not to use the solution with that lens case to determine the actual role of the contact lens disinfecting solution itself in disinfecting the lens cases and, also, to allow better direct comparison with the Polyquat/Aldox solutions. However, this did result in the AQuify solution being used not as recommended by the manufacturer. Participants were advised to discard any solution left in the case after removal of lenses, rinse case with fresh solution (and to never to use water in cases), and air dry (position not specified) cases between disinfection cycles. Participants were all instructed to use the cases for 1 month, return the case to the clinic (or dispose of the case), and use a new case each month. As each participant had scheduled visits at 1 month and 3 months of lens wear, two lens cases were collected for each lens/solution combination for each participant. The cases that were collected usually contained a small amount of disinfecting solution as there was not sufficient time for cases to air dry after lens removal before participants attended the clinic; this was discarded before swabbing the case. After collection, the cases were transferred to the Institute's microbiology laboratory for analysis.
Microbial Analysis of Contact Lens Cases
Lens cases were delivered to the microbiology laboratory within 1 h of collection. At the laboratory, any solution that was contained in the cases was discarded, and the lids and wells (inner well of basket that holds the lenses for ClearCare cases) were swabbed using an alginate swab soaked in phosphate-buffered saline containing 1% (wt/vol) sodium hexametaphosphate to dissolve the alginate. After mixing for 30 s, the solution was cultured as described previously25 by plating aliquots onto three “chocolate” agar plates and one Sabouraud dextrose agar plate (Oxoid, Basingstoke, United Kingdom). One of the three chocolate agar plates was incubated at 37°C in 5% CO2 (2 days incubation), one in aerobic conditions (2 days), and one under anaerobic conditions (4 days). Sabouraud dextrose agar was incubated at 25°C for 7 days to culture for yeast and molds. The number of colony forming units was calculated for each microbial type. Bacteria were identified by Gram stain. Further identification was conducted using a combination of API strips (bioMerieux, Marcy l'Etoile, France) and biochemical tests. Fungi (yeasts and molds) were identified by morphologies of their colonies on agar plates and their conidia. An aliquot from the buffered saline was also plated onto an agar plate seeded with Escherichia coli26 and incubated for 5 days at 37°C. The number of track forming units was then counted as a measure of the number of Acanthamoeba from each case. A pilot study, using 10 randomly chosen flat cases, was performed to determine whether there were differences in contamination rate or types of microbes isolated from the lid vs. the well of each case.
Overall contamination rates, expressed as a binary variable, and contamination rates for type of organism were compared between solution types after adjusting for the two study visits. Data were analyzed using logistic regression with robust estimate of variance to adjust for repeated observations of a participant if they participated in more than one lens/solution combination. Analysis was also performed after assigning different microbes to significant or nonsignificant levels. Table 2 shows the significance level (i.e., number of colony forming units at which a microbe becomes a significant contaminant). The significance levels were estimated based on the assumed pathogenicity of the microbe type and the median number found per contact lens well/basket. Contamination levels based on colony forming units were compared between the solution types after adjusting for visits using linear mixed models and after accounting for within participant correlation. Post hoc multiple comparisons were adjusted using Bonferroni correction, and p ≤ 0.05 was considered statistically significant.
Table 3 details the total number of lens cases analyzed during the course of the study. There were slightly less lens cases analyzed than might be expected (n = 464) because some participants did not return cases at a scheduled visit/appointment. There were no significant differences in the rate or type of microbe isolated from cases in which different lenses had been used (i.e., OASYS/ClearCare vs. Biofinity/ClearCare; PureVision/AQuify vs. Biofinity/AQuify). There were no differences in the rate of contamination or types of microbes contaminating the lids or wells of the lens cases from the pilot study (Table 4), therefore, subsequently, a single swab was used to sample the well and lid at the same time. Similarly, there was no difference in contamination rate or type of microbes between the “left” and “right” hand sides of any lens case, and the data collected from each side were averaged. There were no differences in rate of contamination (Table 5) or types of microbes between lens cases collected and used for 1 month compared with those collected at the 3-month visits but used for 1 month, and so, similarly, these data were be combined.
Overall, 85% of lens cases were contaminated (1 or more colony forming unit per case of any microbial type cultured on agar plates), with cases that had been exposed to AQuify solution being most commonly contaminated (92%) and cases exposed to Opti-Free Express being least commonly contaminated (76%). Fig. 1 shows the rate of contamination in cases exposed to each solution type. Acanthamoeba sp. were never cultured from cases. There were significantly more cases with contamination following the use of AQuify solution compared with either ClearCare (81%; p = 0.05) or Opti-Free Express (76%; p = 0.02; Fig. 1A). There were also differences in rate of contamination of lens cases for different groups of microbes. For any amount of Gram-negative bacteria in a case, cases exposed to Opti-Free Express were less contaminated (7%) than those exposed to AQuify (29%; p = 0.001) or Opti-Free RepleniSH (45%; p = 0.0001; Fig. 1B). Cases exposed to ClearCare were less frequently contaminated (20%) by any level of Gram-negative bacteria than those exposed to RepleniSH (p = 0.01; Fig. 1B). When significant levels of either any microbe or only significant levels of Gram-negative bacteria were compared, cases exposed to Opti-Free Express were less frequently contaminated than those exposed to any of the other three solutions (Fig. 1C, D).
There were differences in the number of colony forming units in total (i.e., for any microbe) for microbes that were cultured from cases exposed to difference solutions (Table 6). These differences were largely because of significant differences in the number of Gram-negative bacteria that were cultured. Cases exposed to Opti-Free RepleniSH had higher numbers of Gram-negative bacteria than those exposed to any other solution (Table 6; p = 0.0001), and the general trend for numbers of colony forming Gram-negative bacteria cultured from cases was Opti-Free RepleniSH > AQuify > ClearCare > Opti-Free Express.
For individual microbial types, genera, or species, there were certain significant differences noted in the rate of contamination of other microbial types (Fig. 2) with certain solutions, namely Staphylococcus aureus: AQuify (6%) = ClearCare (9%) > Express (0%) = RepleniSH (0%; p = 0.008); Staphylococcus epidermidis: Express (32%) < AQuify (56%; p = 0.001) or ClearCare (61%; p = 0.0001), RepleniSH (43%) < ClearCare (61%; p = 0.031); Staphylococcus saprophyticus: Express (18%) < AQuify (45%; p = 0.0001) or ClearCare (44%; p = 0.0001); “viridans” streptococci: AQuify (3%) < Express (13%; p = 0.012); Delftia acidovorans: RepleniSH (26%) > Express (3%; p = 0.017) or ClearCare (1%; p = 0.0001); Serratia marcescens: Express (0%) < AQuify (5%; p = 0.033) or RepleniSH (5%; p = 0.033); Stentotrophomonas maltophilia: ClearCare (2%) < AQuify (11%; p = 0.01) or RepleniSH (14%; p = 0.006); Achromobacter group A: RepleniSH (10%) > ClearCare (1%), AQuify (0%) or Express (0%; p = 0.001); Yeasts: AQuify (9%) = ClearCare (8%) > Express (0%) = RepleniSH (0%; p = 0.006). However, there were no significant differences in the rate of contamination of cases by Bacillus sp. (average rate of contamination over the different solutions = 22%), Corynebacterium sp. (6%), Micrococcus sp. (16%), Propionibacterium sp. (60%), Nocardia sp. (1%), nonhemolytic streptococci (1%), Staphylococcus lugdunensis (2%), Staphylococcus hyicus (6%), other streptococci (2%), Streptococcus pneumoniae (2%), Acinetobacter sp. (1%), Aeromonas hydrophila (<1%), Burkholderia cepacia (1%), Enterobacter cloacae (2%), Klebsiella oxytoca (3%), Pseudomonas aeruginosa (<1%), Serratia liquefaciens (3%), Pseudomonas putida (2%), Moraxella sp. (<1%), Klebsiella pneumoniae (<1%), or Fungi (14%).
This study examined the rate of contamination and the types of microbes that contaminated lens cases during use with three different silicone hydrogel lenses and four different contact lens disinfecting and cleaning solutions. The overall rate of contamination was 85%, which matches closely with rates published previously from studies from the US19 and New Zealand.20 However, the current study is the first to report on rates of contamination of cases used in conjunction with silicone hydrogel lenses and the newer versions of contact lens disinfecting solutions.
One of the significant findings of this study was that use of Opti-Free Express resulted in a significant reduction in colonization frequency of cases by Gram-negative bacteria compared with the other three disinfecting systems. This was surprising given the close similarities between the ingredients of Opti-Free Express and Opti-Free RepleniSH (Table 1). As the two solutions contain the same disinfectants at the same concentrations [polyquaternium-1 (0.001%) and myristamidopropyl dimethylamine (0.0005%)], the differences in anti-Gram-negative activity in lens cases during use could be because of changes in other components. Opti-Free Express contains sorbitol, sodium ethylenediaminetriacetic acid (NaEDTA), and amino-2-methyl-1-propanol, whereas Opti-Free RepleniSH does not appear to contain these components (it contains nonanoyl ethylenediaminetriacetic acid). NaEDTA is known to act as an antimicrobial; it chelates Ca2+ and Mg2+ and can remove these from the outer membrane of Gram-negative bacteria resulting in destabilization of the membrane and bacterial death.27,28 The antimicrobial activity of nonanoyl-EDTA is much less well studied. Neither sorbitol nor amino-2-methyl-1-propanol is known to be antimicrobial. These differences in the ingredients of the two polyquaternium/myristamidopropyl dimethylamine containing solutions are also likely to account for the over-representation of D. acidovorans, S. marcescens, S. maltophilia, and Achromobacter group A in cases that were exposed to Opti-Free RepleniSH rather than Opti-Free Express.
In vitro experiments comparing the effectiveness for Opti-Free Express and Opti-Free RepleniSH are not frequent in the literature. Scheuer et al.29 demonstrated that Opti-Free RepleniSH, Opti-Free Express, and AQuify give 1 to 2 log reduction of Fusarium after 30 min exposure, but although Opti-Free Express and AQuify gave 1 to 2 log reduction of Candida albicans after 30 min exposure, Opti-Free RepleniSH gave <1 log reduction at the same time point. On the other hand, Opti-Free RepleniSH and AQuify gave 3 to 4 log reduction for S. aureus, whereas Opti-Free Express gave <3 log reduction after 30 min, and for Gram-negative bacteria, Opti-Free RepleniSH and Opti-Free Express tended to behave similarly under the short-term (30 min) exposure used.29 Using 24 h of incubation of solutions with Acuvue 2 contact lenses before exposure to Fusarium sp., Opti-Free RepleniSH was reported to retain 4 log reduction in fungal numbers, whereas Opti-Free Express gave a slightly reduced reduction of 3.2 logs.30 This study showed that the rate of contamination of cases by fungal species or the number of fungal cells in cases exposed to Opti-Free Express and Opti-Free RepleniSH were similar and would suggest that these small differences in in vitro activity against Fusarium do not affect in vivo performance.
The cases exposed to hydrogen peroxide (the ClearCare solution) were generally as frequently colonized by bacteria and fungi as the cases exposed to the polyquaternium/myristamidopropyl dimethylamine (Opti-Free Express and Opti-Free RepleniSH) or PHMB (AQuify). This is in contrast to a previously published comparison that demonstrated less frequent contamination of lens cases of users of a different hydrogen peroxide solution compared with users of other disinfection systems.17 Furthermore, in vitro formed biofilms of P. aeruginosa in contact lens cases have been reported to be more effectively killed by hydrogen peroxide solutions compared with those containing polyquaterium-1 or PHMB.31In vitro comparisons of the activity of ClearCare with other nonhydrogen peroxide systems have shown that Opti-Free Express and ClearCare have similar levels of activity against S. aureus, P. aeruginosa, S. marcescens, and Candida albicans, but that ClearCare was less active (2.2 log reduction in microbe numbers) against Fusarium compared with Opti-Free Express (4.2 log reduction).32 In the current study, the rate of contamination of cases by fungi was similar between ClearCare and Opti-Free Express (both 17%), but there were differences in the contamination rate by S. aureus (9% vs. 0%; Fig. 2) and yeasts (8% vs. 0%). In an attempt to better mimic the situation in vivo, Szczotka-Flynn et al.33 tested the effectiveness of AQuify, Opti-Free Express, and ClearCare to kill bacteria that had been allowed to form biofilms on the surface of lotrafilcon A silicone hydrogel lenses. These researchers found that all the solutions were equally active against planktonic cells. However, AQuify only gave <1 log reduction in cell numbers against biofilms of all bacteria genera; Opti-Free Express gave >7 log reduction for P. aeruginosa and S. aureus but only approximately 1 log reduction for S. marcescens; and the ClearCare solution gave >7 log reduction with P. aeruginosa, ∼6.6 log reduction for S. marcescens, and ∼4 log reduction for S. aureus, when they were grown as biofilms. In the current study, the rate of contamination of cases by P. aeruginosa was not different between AQuify, Opti-Free Express, and ClearCare and the rate of contamination of cases by S. marcescens (AQuify = 5%; Opti-Free Express = 0%; ClearCare = 1%) did not correlate with the effects of the different solutions on the biofilm bacteria in vitro. However, the rate of contamination of cases by S. aureus (AQuify = 6%; Opti-Free Express = 0%; ClearCare = 9%) did approximately (negatively) correlate with the effect on the biofilm bacteria (AQuify = <1 log; Opti-Free Express = >7 log; ClearCare = 4 log).
It is possible that the general lack of correlation might be because of the use of additional lens types in the current in vivo study compared with only lotrafilcon A in the study by Szczotka-Flynn et al.33 However, the lack of any significant effect of lens material (balafilcon A, senofilcon A, and comfilcon A) on contamination of cases in this study would argue against a strong effect. Another area that has received some research attention is the ability of certain contact lenses to reduce the effectiveness of disinfecting solutions, most likely because of adsorption of active ingredients into the lens. Rosenthal et al.30 showed that soaking Acuvue 2 FDA group IV lenses for 24 h in solutions containing Alexidine or PHMB reduced anti-Fusarium activity by 50 to 100% but had <20% reduction of activity of polyquaterium-1/ myristamidopropyl dimethylamine containing solutions (e.g., Opti-Free Express or Opti-Free RepleniSH). Ide et al.34 showed that residual antifungal activity of at least 1-log reduction against Fusarium oxysporum was maintained for RepleniSH or solutions containing PHMB or Alexidine at 24-, 48-, and 72-h exposure to senofilcon A (Acuvue Oasys) or balafilcon A (PureVision) lenses. Reduced antifungal activity was shown for senofilcon A and balafilcon A lenses on day 8, when the lenses were paired with a PHMB-containing solution but not with the RepleniSH. Dannelly and Waworuntu35 demonstrated that incubating Acuvue 2 lenses for 1 week in Opti-Free Express or solutions containing PHMB resulted in loss of activity against P. aeruginosa, S. aureus, or Fusarium solani of the PHMB solutions, whereas Opti-Free Express retained its antimicrobial activity. Thus, these data suggest that solutions containing polyquaterium-1/myristamidopropyl dimethylamine are less likely to show reductions in activity when used with a variety of lens types. The lack of effect of the lens materials used in the current study on the contamination rate of lens cases, coupled with a lack of epidemiologic evidence showing differences in rates of microbial keratitis in daily wear with different lens types [soft HEMA-based and Silicone hydrogels (including balafilcon A and lotrafilcon B and A lenses)],14 or indeed an increase level of microbial keratitis with silicone hydrogel lenses (including lotrafilcon A and B and balafilcon A) when worn on a daily wear basis compared with daily wear of HEMA-based lenses (including Acuvue 2 lenses),6 suggest that the uptake of actives (polyhexanide or polyquaterium-1, etc.) into lenses, at least as modeled in vitro, is not a relevant measure of the effectiveness of solutions.
Carnt et al.36 have reported on the rates of adverse responses associated with the participants and lens/solution combinations from the current study. The rate of corneal infiltrative events, those most frequently associated with microbial contamination of lenses,25,37,38 was highest for Opti-Free RepleniSH compared with ClearCare or Opti-Free Express, and corneal infiltrative rate using AQuify was higher than for ClearCare (overall corneal infiltrate rates RepleniSH > AQuify > Express > ClearCare).36 These data show good correlation with the data on lens case contamination published in this study (contamination with RepleniSH > AQuify > Express), apart from with ClearCare where the level of microbial contamination of lens cases was equivalent to that with RepleniSH. Perhaps other factors, such as transfer of microbes from the case to the lens and the viability of those microbes, are important.39 There may be effects of multiple species being present in the case on the disinfecting solutions efficacy. Certainly co-incubation of P. aeruginosa with Acanthamoeba sp. can alter susceptibility of the latter to disinfection.40 In the case of hydrogen peroxide solutions, the presence of catalase-producing microbes, such as the Staphylococci, may afford some protection to other microbes. Also, differences in disinfecting time (solutions are often tested in vitro at the manufacturers recommended minimum time but may be used in vivo for longer periods) may also explain the noticeable lack of correlations between in vitro and in vivo performance of the solutions; indeed, the ClearCare solution case contains an inbuilt platinum catalyst that degrades the hydrogen peroxide to water over time, whereas the Polyquad/Aldox and PHMB solutions will continue to have disinfecting activity.
There are now available contact lens cases impregnated with silver, which have been reported to provide extra antimicrobial activity. These lens cases are available from three manufacturers, CIBA Vision; Sauflon Pharmaceuticals, Twickenham, United Kingdom; and Marietta Vision, GA. Indeed, the AQuify solution used in this study is sold with the CIBA Vision silver lens case (although that case was not used in the current study). Amos and George41 have reported that use of the CIBA Vision silver lens case (variously termed MicroBlock or ProGuard cases depending on geographical area) resulted in only 26% of cases being contaminated, whereas 67% of nonsilver cases were contaminated. Although some of the lens case types and microbial culturing conditions were different between the current study and that reported by Amos and George,41 which may account for the slight reduction in case contamination rate of nonsilver cases, the dramatic reduction in contamination rate using silver-impregnated lens cases is very encouraging. It would be intriguing to examine whether use of Opti-Free Express, the solution least likely to encourage contamination of cases in the current study, with these silver lens cases can further reduce contamination rates.
It is the function of all the contact lens disinfecting solutions to reduce the contamination rate of contact lenses. Ultimately, this reduction in contamination of lenses is likely to result in a reduction in microbially driven adverse events during lens wear. The current study suggests that use of a particular Polyquat/Aldox solution (Opti-Free Express) can result in significantly less colonization by Gram-negative bacteria in contact lens cases. In the future, studies should be designed with sufficient power to test whether changes in case contamination result in changes to adverse responses rates.
We thank the help of all the staff of the biosciences laboratory and clinic at the Institute for Eye Research.
Mark D. P. Willcox
Institute for Eye Research
Rupert Myers Building
University of New South Wales
Sydney, New South Wales 2052
Australia; e-mail: firstname.lastname@example.org
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