For the microbiological analysis, the following organisms were studied: Pseudomonas aeruginosa (ATCC 9027), Serratia marcescens (ATCC 13880), Staphylococcus aureus (ATCC 6538), Candida albicans (ATCC 10231), Fusarium solani (ATCC 36031) and Acanthamoeba castellanii (ATCC 50370). Test solutions studied are given in Table 1.

For the bacteria and fungi, stand-alone (biocidal) and regimen assays were performed according to ISO 14729.10 Briefly, in the stand-alone procedure the test solutions were challenged with 1 × 106/ml organism and the number of survivors determined by culture viability at 0 and 6 hr using the WASP Spiral Plater and ProtoCOL colony counter system.16 At each time point the test samples were first diluted 1:10 into DifcoW Letheen Broth neutralizing broth before determining the number of surviving organisms as colony forming units (cfu).

In the regimen assay, four sets of the following lenses were tested per organism: Soflens® 38 (polymacon, Bausch & Lomb), Acuvue® 2 (etafilcon A, Vistakon), Acuvue® Advance® (galyfilcon, Vistakon), O2Optix®/AirOptix® (lotrafilcon B, CIBA Vision), PureVision® (balafilcon A, Bausch & Lomb) and Biofinity® (comfilcon A, CooperVision) lenses. Although not required by the protocol, organic soil was included in the organism challenge inoculum and comprised a final concentration of 300–3000 heat killed Saccharomyces cerevisiae (ATCC 9763) in 0.003 % heat-inactivated fetal bovine serum. Briefly, four lenses of each test type were inoculated with approximately 2 × 106 organisms and left to adhere for 5 minutes. Each lens was then subjected to a “No Rub but Rinse” with NuMPS using 5 seconds rinse per side followed by a 6 hour soaking time in 3 ml of the solution in a contact lens storage cases (ABS/polypropylene, Abbott Medical Optics Inc.). At the end of the regimen procedure, each test lens and soaking solution was neutralized in Difco™ Letheen Broth followed by filtration and culture of the lens and membrane to determine the presence of surviving organisms (cfu).

Acanthamoeba trophozoite and cyst biocidal and regimen assays were performed as described previously.5,17,18 For the regimen assay, lenses were inoculated with 4 × 104 trophozoites or cysts per lens and subjected to a “Rub and Rinse” regimen with the NuMPS using 4 seconds rub and 5 seconds rinse per side, followed by a 6 hour soaking time in 3 ml of the solution in a contact lens storage cases (ABS/polypropylene, Abbott Medical Optics Inc.). Remaining viable Acanthamoeba on the lenses and in the soak solution were determined by culture assay as described previously.5

The capacity for the test solutions to induce Acanthamoeba trophozoite encystment during incubation in the solutions was performed according to the method described by Kilvington et al. using Complete® MoisturePlus® MPS as the positive control solution. This MPS formulation has previously been shown to induce significant trophozoite encystment.19

Loss of MPS antimicrobial efficacy following partial evaporation was also studied. The solutions were evaporated under a stream of air to 2× and 4× concentration by weight, challenged with F. solani or A. castellanii trophozoites and the level of organism kill determined after 6 hr contact time.

In vitro cytotoxicity potential of NuMPS and MPS-3 (OPTI-FREE® RepleniSH® Multi-Purpose Solution) was evaluated according to USP < 87 > and ISO 10993-5.11,20,21 Five soft contact lens types were studied: Soflens® 38 (polymacon, Bausch & Lomb), Acuvue Advance® (galyfilcon A, Vistakon), O2Optix® (lotrafilcon B, CIBA Vision), PureVision® (balafilcon A, Bausch & Lomb) and Biofinity® (comfilcon A, CooperVision) lenses. Briefly, the lenses were soaked in 100 ml of the test solutions, in triplicate, for four days. Confluent monolayers of L929 mouse fibroblasts were then exposed to treated contact lenses for 24 hours and scored for reactivity according to USP Direct Contact Test criteria (Table 2).21 Polypropylene pellets and latex rubber served as negative and positive controls, respectively.

A three-month, double-masked, parallel group clinical trial comparing the safety and acceptability (comfort and lens cleanliness) of the NuMPS (test) to MPS-3 (control) was conducted with four types of silicone hydrogel contact lenses: Acuvue® Advance® (galyfilcon A, Vistakon), PureVision® (balafilcon A, Bausch & Lomb), O2Optix® (lotrafilcon B, CIBA Vision), Biofinity® (comfilcon A, CooperVision) and a FDA Group IV material (e.g., Acuvue® 2, [etafilcon A Vistakon]). Subjects were enrolled in a 2:1 ratio (test vs. control) for each lens material. Of the 270 subjects enrolled, 177 were assigned to the test regimen and 93 to the control. In the test group the subjects used a “Rub and Rinse” regimen in their lens care whereas for the control a “No Rub but Rinse” regimen was used. Safety was primarily assessed through slit-lamp observations and the occurrence of adverse events (e.g. keratitis, hypersensitivity or visual acuity loss).22 All slit lamp evaluation findings were graded on a 0 to 4 scale, where 0 = None, 1 = Trace, 2 = Mild, 3 = Moderate, and 4 = Severe. Findings were categorized as: corneal edema, corneal neovascularization, corneal staining, bulbar hyperemia, palpebral conjunctiva and other findings. Acceptability was assessed via lens cleanliness (lens deposition as described below) and lens wearing comfort rated on a scale from 0–10 (0 = intolerable and 10 = cannot feel lens) compared to baseline assessments.

Lenses worn in a daily mode using either the test (NuMPS) or control multipurpose solution (MPS-3: see above section, Clinical investigation) were assessed at the end of a wear period (2 wks or 30 days, depending on lens type): n > 30/15 for test/control lenses for protein (left lens) or lipid (right lens) analysis for each lens type tested in the clinical investigation (see above section). All involved parties (including the analyst) were masked regarding control and test lenses. Protein analysis was by a modified Lowry technique utilizing acetonitrile:water (1:1) containing 0.1 % trifluoroacetic acid to extract protein from lenses.23 Lipid analysis involved extracting lipids from lenses with toluene:isopropanol (4:1), followed by deposition of the extracted lipids on a Teflon circular membrane (6 mm diameter) in a well of a 96-well plate (glass) by air evaporation.24,25 Mid-infrared transmission analysis of the Teflon membrane with deposited lipids, based upon functional groups specific to lipid types, was done using a Nicolet Avatar-370 Fourier Transform Infrared Spectrometer. Glyceryl tristearate (99 %, Aldrich, USA) was used to prepare standards for the analysis.

Cochran-Mantel-Haenszel test was used to compare difference in overall corneal staining between the two regimens and Fisher's exact test for adverse events. General Linear Model analysis was used to analyze acceptability measures for overall cleanliness and lens wearing comfort. Analysis of protein and lipid deposition on lenses was performed using Wilcoxon rank-sum test.

The log10 reduction in bacterial and fungal viability 6 hours exposure time to the test solutions is shown in Table 3. With the exception of MPS-3, which gave a 2.3 log10 kill for S. aureus, all commercial MPS and Perox gave > 4.0 log10 reduction in bacterial viability. A greater range in efficacy was observed with the fungi. For C. albicans, MPS-1 and MPS-4 gave 1.0–1.1 log10 kill respectively and > 3.0 log10 for the other solutions, including Perox. For F. solani, a 1.7–2.1 log10 reduction was obtained for MPS-4 and MPS-3, respectively, and > 3.0 log10 for all other commercial solutions. NuMPS showed > 4.0 and > 3.0 log10 reduction for all bacteria and fungi at 6 hours, respectively.

The results for the Acanthamoeba biocidal studies are shown in Table 4. All the solutions showed activity against trophozoites, ranging from 2.2–3.6 log10 kill after 6 hours. The results for the cysts were more variable and ranged from 0.1–2.4 log10 for MPS-3 and Perox, respectively, and > 3.0 log10 kill for the others.

With the regimen testing, the NuMPS gave < 10 cfu surviving bacteria and fungi on the lenses or in the soaking solution under No Rub but Rinse conditions and < 10 viable Acanthanmoeba trophozoites or cysts when Rub and Rinse was used.

No evidence for trophozoite encystment (0 %–5 %) was observed during 24 hr incubation in any of the test or NuMPS solutions. The control encystment solution of Complete® MoisturePlus® MPS gave 30 % encystment.

The efficacy of test solutions against F. solani and A. castellanii trophozoites after evaporation to 2× and 4× concentration and a 6 hr disinfection time is shown in Table 5. For F. solani, MPS-5 and MPS-3 lost 85 %–98 % and 70 %–85 % of activity at 2× and 4× concentration, respectively. No loss in efficacy on concentration was found with MPS-2 or NuMPS. For A. castellanii trophozoites, MPS-5 showed a 13 %–51 % reduction in efficacy and MPS-3 a 38 %–9 % loss at 2× concentration and 4×, respectively. MPS-2 gave a 14 % loss in activity at 2× concentration and 0 % at 4×. No loss in trophozoite efficacy on concentration was found with NuMPS.

In vitro cytotoxicity results are described in Table 6. Here NuMPS showed grade 1 (slight) with PureVision® and grade 2 (mild) with Acuvue Advance® and O2Optix® lenses, compared to MPS-3 which exhibited a grade 3 (moderate) for these SiHy lenses. Both solutions scored a grade 2 for Soflens® 38 lenses and grade 3 for Biofinity® lenses.

The overall results indicated no significant difference in corneal staining between the two regimens. Corneal staining incidence at Day 90 was 18 % trace and 2.9 % mild with NuMPS; and 11.9 % trace, 3.6 % mild, and 1.2 % moderate with MPS-3 (p > 0.05). However, the incidence of adverse events in control subjects was significantly greater than the test subjects with 11.8 % (11/93) versus 2.8 % (5/177), respectively (p < 0.05). These adverse events were not specific to any one lens type, but were more often inflammatory (keratitis, conjunctivitis) or hypersensitivity responses (solution sensitivity, redness, tearing, etc.) with no cases of confirmed microbial infection. With regard to acceptability measures, there was no significant difference between the two regimens among all lenses tested in terms of overall cleanliness and lens wearing comfort, with lens wearing comfort mean ratings in the test and control groups of 8.7 and 8.6, respectively (p > 0.05).

The conventional etafilcon A (Acuvue 2®) worn lenses were found to contain much higher levels of protein than any of the four silicone hydrogel types, with average protein levels of 50-60 μg/mg of dry lens material (Table 7). Although the average protein for this lens type (FDA Group IV) slightly favored the control solution (MPS-3), it was not statistically significant (p = 0.10). All four silicone hydrogel types showed an average of less than 1 μg protein per mg of dry lens material for lenses used with either the control (MPS-3) or test solution (NuMPS). No statistically significant difference was found between the solutions regarding protein deposition on three of the silicone hydrogel lens types (p = 0.70–0.97). However, lotrafilcon B lenses (O2Optix®) exhibited a significantly lower level of protein deposition with NuMPS (0.04 μg/mg of lens) compared to the control MPS-3 (0.12 μg/mg, p = 0.01).

As shown in Tables 8-9, lipid levels were significantly higher for all worn silicone hydrogel lenses (1-5 μg/mg of dry lens material, depending on lens type) than for the conventional etafilcon A (Acuvue 2®) lens type (0.4 μg/mg of lens). Of the silicone hydrogel lenses, galyfilcon A (Acuvue Advance®) showed the highest total lipid deposition (∼4.0 μg/mg) and lotrafilcon B (O2Optix®) exhibited the lowest (∼1.0 μg/mg). There were no statistically significant differences in the amount of lipid deposition between the test and control solutions, although balafilcon A lenses (PureVision®) deposited less ester-type lipids (such as aliphatic glyceride esters) with NuMPS than MPS-3 with data tending toward significance (p = 0.06). Overall, the average amount of total lipid deposition for all lenses was identical for the two products and averaged 2.2 μg of lipid per mg of lens.