As many as 43 percent of healthy individuals may have Staphylococcus aureus in and around their eyes, and it's not unusual to isolate this organism from patients who have microbial keratitis (Silbert, 1988). In fact, one study (Alexandrakis, 2000) has suggested an increase in the rate of S aureus-related bacterial keratitis.

The U.S. Food and Drug Administration (FDA) and the International Standards Organization (ISO) have outlined specific procedures to evaluate the inherent microbiocidal activity of a disinfecting solution using a stand-alone test (Rosenthal, 2002). The stand-alone method provides a quantitative measure by which researchers evaluate disinfecting solutions against FDA- and ISO-established performance criteria, and it may be used to assess the relative antimicrobial efficacy of different disinfecting solutions. ISO identifies a minimum standard for primary efficacy of not less than a 3-log reduction for bacteria (S aureus ATCC 6538, Pseudomonas aeruginosa ATCC 9027, Serratia marcescens ATCC 13880) and a 1-log reduction for mold (Fusarium solani ATCC 36031) and yeast (Candida albicans ATCC 10231) in the minimum disinfecting time as specified according to the manufacturer's label.

During their development phase, researchers assess all contact lens disinfectant products against FDA and ISO requirements. Although all marketed lens care products have passed this criteria on the laboratory strains of bacteria and fungi (Rosenthal, 2002), low levels of infections still occur in lens wearers. Alexandrakis (2000) even reports what appears to be an increasing rate of antibiotic resistance among S aureus isolates from patients who have bacterial keratitis. This suggests that the ISO guideline as a reference standard may not predict outcomes in real-world use situations. Testing disinfectant products against additional clinical strains may assist in evaluating these products.

Patients can easily self-transmit bacteria that may cause infections from the skin to the eye while handling contact lenses and lens cases. Studies have shown that the degree of contamination may be related to user compliance with product instruction (Sven, 1994; de Andrade Sobrinho, 2003; Khor, 2006). The use of a contact lens disinfectant product that has a high level of antimicrobial activity may provide additional assurance that contaminating microbial levels will be effectively reduced.

We performed the investigation reported here to determine the relative biocidal efficacy of several multipurpose solutions against the ISO reference strain of S aureus. Researchers isolated the additional strains tested from lens cases, solutions, lenses and/or swabs of the cul-de-sac area of the eye of asymptomatic contact lens wearers.

Test Solutions We included six multipurpose lens care solutions in this study. Four of the products use the disinfecting agent known as polyhexamethylene biguanide (PHMB); these included ReNu MultiPlus (Bausch & Lomb), ReNu Multi-Purpose Solution (B&L), Complete MoisturePlus (Advanced Medical Optics) and Aquify (CIBA Vision). The remaining two products, Opti-Free Express (Alcon) and Opti-Free Replenish (Alcon) utilize the disinfecting agent known as Polyquaternium-1.

Test Organisms We used Staphylococcus aureus ATCC strain 6538 as a reference. We collected other S aureus strains clinically as isolates, obtained in a frozen state from Nelson Laboratories (Utah) as a part of a study conducted by B&L on asymptomatic contact lens wearers of Japanese descent. Most of these subjects resided on the U.S. West coast or in Hawaii. Researchers obtained 21 isolates originating from 7 subjects from either contact lens case solutions, contact lenses or from a swab of the cul-de-sac area of the eye. All isolates were grown out on chocolate agar or blood agar, identified and preserved at ultra-low temperatures.

Test Procedure We cultured all clinically collected isolates and the ATCC standard in soybean casein digest agar medium, USP for 18 to 24 hours, and then washed them via centrifugation at no more than 4000 x g for a maximum of 10 minutes at 20�C to 25�C. Organisms were resuspended in Dulbecco's Phosphate Buffered Saline plus 0.05% w/v polysorbate 80 (DPBST) or the equivalent, centrifuged a second time and resuspended in DPBST at 1 � 10⁷ to 1 � 10⁸ CFU/mL, as determined spectrophotometrically at a wavelength of 490nm.

We aliquoted a minimum of 10mL of each test solution into a separate test tube for each organism undergoing testing and inoculated them with a suspension of organism sufficient to provide a final concentration of 1.0 � 10⁵ to 1.0 � 10⁶ CFU/mL. The volume of the inoculum did not exceed 1 percent of the sample. We vortexed samples to ensure adequate dispersion and stored them at 20�C to 25�C. We took 1mL aliquots of test solution for viability counts at 100 percent of the minimum recommended disinfection time for all test solutions. Samples were serially diluted in an appropriate neutralizing broth and aliquots were vortexed vigorously, then plated onto recovery agar plates in triplicate. We incubated recovery plates at 30�C to 35�C for two to four days.

Figure 1 shows that all the multipurpose solutions met the primary criteria for a stand-alone disinfectant, defined as a minimum 3-log reduction in bacterial colonization as set forth in the ISO 14729 standard for stand-alone contact lens care products, when tested against the standard ATCC strain of S aureus. Figure 2 shows the results of each solution's performance against bacterial strains found in the environment and as a part of normal eye flora.

Figure 1. All tested solutions met the primary criteria for stand-alone disinfection.

Figure 2. Solution effectiveness against environmental and eye flora bacterial strains.

ReNu MultiPlus maintained a high rate of disinfection of at least a 4.5-log reduction in bacterial colonization for all isolates tested, with an average of 4.8 logs. The other PHMB-containing solutions, ReNu Multi-Purpose Solution, Complete MoisturePlus and Aquify, far surpassed the required 3.0-log reduction for the required organisms and had equally high performance against all clinically collected isolates tested, with average reductions of 4.5, 4.7 and 4.7 logs, respectively.

In comparison for the polyquaternium-1-containing solutions, Opti-Free Express had less than a 3-log reduction for six of the 11 clinically collected isolates tested, with an average log reduction of 3.1 (ranging from 1.9 logs to 3.9 logs). Opti-Free Replenish demonstrated the weakest activity against the clinical S aureus strains, with an average 2.2-log reduction in bacterial colonization (ranging from 1.6 logs to 3.1 logs).

Contact lenses and lens care products are exposed to many potential sources of microbial contamination. Microorganisms from the environment, including those found in or on water, air, soil, animals and plants, as well as the normal flora of the eye, may, under certain circumstances, result in infection (Rosenthal, 2000).

Some reports have suggested an increased incidence of multiple antibiotic-resistant strains of S aureus, especially among hospital patients (Tabbara, 1989; Alexandrakis, 2000).

ReNu MultiPlus, ReNu Multi-Purpose Solution, Complete MoisturePlus and Aquify demonstrated efficacy that far exceeded the minimum acceptance criteria of 3-log reduction for the required strain and demonstrated an equally impressive kill of the clinically collected strains.

Although Opti-Free Express met the ISO 14729 standard for biocidal activity when tested against a laboratory standard strain of S aureus, it didn't consistently maintain this level of activity when tested against a potentially more relevant scenario of isolates obtained from asymptomatic contact lens wearers. The average log reduction of bacterial levels for Opti-Free Express was 3.1, with results of less than a 3-log reduction in more than half of the clinically collected isolates tested. Opti-Free Replenish demonstrated even weaker biocidal activity when tested against the clinically collected isolates, with an average log reduction of 2.2.

As the effectiveness of contact lens care products comes under closer scrutiny, it becomes more evident that these solutions should be tested under more clinically relevant conditions. When we tested stand-alone biocidal efficacy of ReNu MultiPlus against S aureus obtained as clinically collected isolates from asymptomatic contact lens wearers, this solution demonstrated high levels of antimicrobial activity. We observed consistently high efficacy for the other PHMB-containing solutions tested, including ReNu Multi-Purpose Solution, Complete MoisturePlus and Aquify. Of the Polyquaternium-1-containing disinfecting solutions, Opti-Free Express demonstrated an efficacy which averaged just over a 3-log reduction against the clinical S aureus strains (3.1 log-reduction average), with less than a 3-log reduction for more than half of these clinical strains. Opti-Free Replenish demonstrated only an average 2.2-log reduction. These results suggest that PHMB-containing solutions, such as ReNu MultiPlus, may provide a greater disinfection advantage against environmental strains of this problematic microorganism.