2.1

Contact lens materials

Three cosmetic hydrogel materials were used in this study: etafilcon (Acuvue Define, Johnson & Johnson, New Brunswick, NJ, USA), nelfilcon (Freshlook Colorblends, Alcon, Fort Worth, TX, USA), and hilafilcon (Naturelle, Bausch & Lomb, Bridgewater, NJ, USA). Lens specifications according to manufacturers’ product information are shown in Table 1 .

2.2

Participants and wearing schedules

Volunteers willing to wear the provided daily disposable contact lenses for at least 8 h were recruited into the study. Inclusion criteria included: (1) age 20–35 years; (2) myopia less than -6.00 D and astigmatism less than -1.50 D; and (3) previous soft contact lens wear discontinued for at least 2 weeks. Exclusion criteria included: (1) previous rigid gas permeable lens or orthokeratology wear; (2) any ocular disease requiring topical medication; (3) any systemic disease that may affect the ocular surface; and (4) pregnancy. The aim and purpose of this study were thoroughly explained and written informed consent obtained from all volunteers. This study was approved by the relevant Institutional Review Board and adhered to the tenets of the Declaration of Helsinki.

Each volunteer wore one type of cosCL for 21 days in both eyes, then stopped all contact lens wear for 14 days (wash out period) before wearing the next type of cosCL for 21 days. The subjects repeated the process until all three types of cosCLs had been worn. The order of cosCL worn by each subject was randomized. These experienced contact lens wearers were instructed to wash and dry their hands before insertion of the lenses each morning. After wear each day, the contact lenses were collected aseptically by trained staff, then placed in sterile containers. All used contact lenses were checked for completeness of lens with no tears or chipped pieces. The used lenses were then subjected to experiments involving microbial identification, P. aeruginosa adhesion tests, and disinfection solution sensitivity tests.

2.3

Identification of bacteria colonization to worn contact lenses

Worn contact lenses were retrieved with aseptic forceps and placed in 1 ml of sterile phosphate buffered saline (PBS) stored in aseptic containers. The lenses were transported to the laboratory for maceration by tissue homogenizer (Polytron Homogenizer PT 4000, Kinematica AG, Luzern, Switzerland) in 1 ml of PBS. Serial dilution was then performed and aliquots of 100 ul were plated onto tryptone soya agar and chocolate agar plates. The plates were incubated aerobically at 37 °C in CO2 incubators for 12 h. Identification of the organisms was done by Gram’s stain, using standard biochemical methods [ ] or colorimetry methods using Vitek2 Compact (BioMerieux, Marcy l’Etoile, France).

2.4

Bacterial strains and culture conditions

Standard strains (PAK, PA103, 6294, 6206) and mutant strains (PAKΔ pscC, courtesy of Stephen Lory, Harvard University, and PA103Δ pscC, courtesy of Timothy L. Yahr, University of Iowa) were stored in frozen stocks until needed. Previously-characterized clinical keratitis isolates (2007AX44, 2007A01, 2007AD46, 2002AP68) were also used. [ ] Frozen stocks were defrosted and loops of bacterial solution were plated onto tryptone soya agar and incubated overnight. The strains were confirmed as P. aeruginosa by green pigment production and positive cytochrome oxidase test (BD Oxidase Reagent Droppers, BD Bioscience, Franklin Lakes, NJ, USA). A single colony was sub-cultured and inoculated into 15 ml of tryptone soya broth supplemented with 1% glycerol, 100 mM monosodium glutamate, and 2 mM EGTA as inducing conditions. The bacteria were harvested by centrifugation at 9600 g for 10 min. The resulting pellet was washed once with 5 ml of normal saline and re-suspended to concentrations as required for experimentation.

2.5

Rub-off test

All three types of unused cosCLs were subjected to a rub-off test, following methods previously described by Chan et al. [ ] In brief, the concave and convex surfaces of unused lenses were rubbed with normal saline-wetted cotton swabs for 20 times each. Pigment dislodgement was identified by observation of transference of color pigment onto the cotton swab tip. If no pigment was found after 20 rubs, the contact lens was labelled as passing the rub-off test. Six lenses of each type were tested.

2.6

Sensitivity of P. aeruginosa to disinfectant solutions

Disinfectant solutions tested in this study included Optifree Replenish (i.e. Replenish, Alcon, Fort Worth, Texas, USA), Optifree Pure Moist (i.e. Pure Moist, Alcon, Fort Worth, Texas, USA), Renu Fresh (i.e. Renu, Bausch & Lomb, Bridgewater, NJ, USA), and AoSept Plus (i.e. AoSept, Ciba Vision, Duluth, GA, USA). The manufacturer’s recommended disinfection times were 6 h for Optifree Replenish, Optifree Pure Moist, and AoSept Plus. Renu Fresh required 4 h for disinfection. Lenses not passing the rub-off test were selected for experimentation. Worn contact lenses were incubated with 1 ml of ∼10 ⁸ bacteria for 2 h, then washed thrice by careful dipping in 3 ml of PBS. The lenses were then transferred to a fresh well (24-well tissue culture plate, Sarstedt, Leicester, UK) and soaked with 1 ml of disinfectant solution at 25%, 50%, 75% and 100% of manufacturer’s suggested disinfection time. Next, the disinfectant solution was removed and 1 ml of Dey-Engley neutralizing broth (Sigma-Aldrich, St. Louis, MO, USA) was added [ , ]. The wells were allowed to sit at room temperature for 15 min. The lenses were then macerated and 0.1 ml aliquots were plated onto tryptic soy agar to determine the quantity of viable bacteria still present on the lenses. The experiments were repeated at least three times and the results averaged.

2.7

Statistical analysis

All data were entered into an Excel data sheet and analyzed using SAS 9.4 (SAS Institute Inc, Cary, NC, USA). Chi square test was used to compare the culture-positive rates and the number of isolates for all three types of cosCLs. Wilcoxon sign rank test was used for non-parametric data analysis. P values less than 0.05 were considered as significant.

3.1

Demographics of participants

A total of 24 female volunteers participated in this study. The average age was 30.6 years (range 28–34 years). All participants had previous experience of wearing hydrogel contact lenses; 11 had previously wore daily disposable hydrogel contact lenses. Another 11 had worn monthly disposable hydrogels and two participants had worn planned disposable hydrogel contact lenses. No participants had worn extended wear contact lenses. Because the test excluded non-working days and national holidays, the total number of retrievable contact lenses was 2400 (816 etafilcon lenses, 768 nelfilcon lenses, and 816 hilafilcon lenses).

3.2

Microbial analysis of worn cosmetic contact lenses (cosCLs)

A total of 1152 contact lenses (384 lenses of each type) were tested consecutively to identify the attached microorganisms. A total of 364 lenses had positive cultures, giving a total culture-positive rate of 31.6%. The culture-positive rates for etafilcon, nelfilcon, and hilafilcon were 21.6%, 33.1%, and 40.1% respectively. Hilafilcon had a significantly higher number of culture-positive lenses than the other two types of lenses (P = 0.0001, chi-square test). Table 2 shows the frequency of isolation of each type of microorganism for each type of contact lens. Of the 376 total isolates found, gram-positive and -negative isolates accounted for 85% and 13% of the total, respectively. Yeast was found in 1.3% of the isolates. The percentage of gram-positive and -negative isolates found on each type of cosCL is shown in Fig. 1 . For both gram-positive and -negative organisms, hilafilcon had the highest number of isolates identified. For all isolates, significantly more were found on hilafilcon lenses than on etafilcon lenses (*P < 0.0001, chi-square test, Fig. 1 C). Although most lenses had one type of microorganism isolated, 9 lenses had more than one type of isolate. The most commonly identified microbe on all cosCLs of asymptomatic lens wearers was coagulase negative staphylococcus (CNS), comprised mainly of commonly found skin commensal bacterium. The next highest identified microbe was Pseudomonas species (sp), accounting for approximately 12% of all identified isolates. The Pseudomonas species included P. aeruginosa , P. flurorescens, P. oryzihabitans, and P. maltophilia (S. maltophilia) . Other non-identified non-fermenting gram-negative bacilli were found mainly on hilafilcon lenses. Taking account of all gram-negative bacilli, hilafilcon lenses generally had a higher likelihood of gram-negative bacilli attachment compared to etafilcon and nelfilcon lenses, although the difference did not reach statistical significance.

Table 2

Identification of microbial organisms on worn cosmetic contact lenses.

Organism	Isolates (N,%)	Etafilcon	Nelfilcon	Hilafilcon
Gram-positive
Staphylococcus coagulase-negative	310	69 (22.3)	112 (36.1)	129 (41.6)
Micrococcus sp	4	0 (0)	2 (50)	2 (50)
Streptococcus sp	3	3 (100)	0 (0)	0 (0)
Bacillus spp.	4	0 (0)	4 (100)	0 (0)
Total	321 (85.4)	72 (22.4)	118 (36.8)	131 (40.8)
Gram-negative
Pseudomonas sp	46	11 (23.9)	14 (30.4)	21 (45.7)
Undetermined Gram-negative bacillus	4	0 (0)	0 (0)	4 (100)
Total	50 (13.3)	11 (22)	14 (28)	25 (50)
Yeast	5	2 (40)	0 (0)	3 (60)
Total	5 (1.3)	2 (40)	0 (0)	3 (60)

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