In Vitro Efficacy of Contact Lens Solutions Against Various Corneal Fungal Isolates

Introduction

Microbial keratitis is a potentially blinding condition that represents the most severe complication related to contact lens wear. In microbial keratitis associated with contact lens use, fungi are uncommon etiologic agents, accounting for less than 5% of all cases.^1–4

Although rare, fungal keratitis is a serious ophthalmic condition, with a guarded prognosis and significant morbidity. ⁵ It requires prolonged topical treatment with antifungal agents, which are toxic, costly, and usually not available commercially, with the exception of natamycin (pimaricin), the only antifungal ophthalmic medication on the market. Prolonged medical therapy may yield a cure or control the disease sufficiently to allow corneal transplantation a chance of success.

Contact lens-related keratitis has been associated with microbial contamination of the contact lens, contact lens solution, and contact lens storage case. ⁶ Overall, contact lens solutions are more effective against bacteria than fungi.^7,8 Antimicrobial performance of disinfecting solutions is important in reducing the microbial contamination of contact lens care systems.

Recent reports of disproportionate outbreaks of Fusarium contact lens-associated keratitis led to the removal of the Bausch & Lomb multipurpose lens care solution ReNu with MoistureLoc^® from the global market in 2006.^9,10 Although an association between Fusarium keratitis and the use of ReNu with MoistureLoc has been found, the specific mechanism for this association is still uncertain. Evidence suggests that exposure to Fusarium is likely to have been the result of extrinsic contamination of contact lens solution bottles or lens cases occurring outside the manufacturing or storage processes, possibly in patients' homes. Poor adherence to contact lens care regimens, such as storing the lenses without emptying and replacing the solution in the case every day, or just adding extra solution to the case (topping off) have been associated with the recent outbreak of Fusarium keratitis. ¹⁰ Although ReNu with MoistureLoc was found to meet all current biocidal standards against Fusarium, persistence of this activity in a variety of environments is not customarily tested and is currently unknown.

Little is known about the efficacy of disinfecting solutions against fungal lens case contamination. Given the frequent failures of medical and surgical treatment, disinfecting solutions effective at killing fungal organisms are important in preventing corneal infection. The purpose of this study was to investigate the susceptibility of different fungal strains belonging to 3 fungal genera frequently implicated in keratitis (Aspergillus, Fusarium, and Curvularia) to 6 commercially available contact lens disinfecting solutions.

Methods

Thirteen filamentous fungal isolates (6 Aspergillus flavus, 3 Aspergillus fumigatus, 3 Fusarium spp., and 1 Curvularia sp.) were used for the present study. All the fungal strains were isolated from eyes with corneal infection at the Institute of Ophthalmology, Joseph Eye Hospital, Tiruchirapalli, India.

Contact lens disinfection solutions available worldwide, containing commonly used active ingredients [e.g., hydrogen peroxide, polyquaternium, polyhexanide (PHMB)] were selected for this study. The following disinfection systems were tested (Table 1): Arion Cronos (Disop, Madrid, Spain), containing 3% hydrogen peroxide+microbial catalase (5,000 IU/tablet); Complete RevitaLens (AMO, Abbott Park, IL), containing polyquaternium-1 0.0003% and alexidine 0.00016%; Dua Elite (Disop), containing polyhexanide (PHMB) 0.0001% and sodium hyaluronate; Opti Free Express (Alcon Laboratories, Fort Worth, TX), containing polyquaternium-1 0.01% and myristamidopropyl dimethylamine (aldox) 0.0005%; Regard (Advanced Eyecare Research Ltd., High Wycombe, United Kingdom), containing the chlorite/peroxide complex; Oxysept Comfort (AMO Ireland, Dublin, Ireland), containing 3% hydrogen peroxide+catalase (0.1 mg/tablet). The 1-step hydrogen peroxide system Oxysept was evaluated both with and without the neutralizer.

Complete RevitaLens, Dua Elite, and Opti Free Express share the same general mechanism of action, that is, disruption of the microbial cell membranes ultimately leading to the organism death. Conversely, Arion Cronos, Regard, and Oxysept Comfort contain oxidizing agents acting on proteins, lipids, and DNA, and causing microbial cell death.

Susceptibility testing experiments were performed at the Department of Biomedical Sciences, Section of Clinical and Experimental Microbiology, University of Sassari, Sassari, Italy.

Before starting susceptibility experiments, all the disinfecting solutions underwent sterility control testing. Briefly, 100 μL of each solution was inoculated on sheep blood and Sabouraud dextrose agar plates, which were then incubated at 37°C and at 30°C for 72 h and 7 days, respectively, to rule out microbial contamination. After incubation, all the solutions examined yielded no bacterial or fungal growth.

Susceptibility testing was performed as follows: all the test isolates were subcultured onto Sabouraud dextrose agar slants and incubated at room temperature for 3 days, after which, conidia were harvested from the cultures using sterile distilled water containing Tween 80. The conidial suspensions were prepared to an optical density equal to 0.5 McFarland standard [∼10⁸ colony-forming units (CFU)/mL]. To confirm the concentrations of the conidia, aliquots (5 μL) of each suspension were removed, serially diluted in sterile phosphate-buffered saline to reach a final concentration of 10² CFU/mL, and then seeded on Sabouraud dextrose agar plates for manual counting of fungal colonies.

Ten microliters of each conidial suspension was added to 990 μL of each test contact lens disinfecting solution so as to achieve a target concentration of 10⁶ CFU of the conidia per mL of the solution. Arion Cronos and Oxysept Comfort, 2 one-step 3% hydrogen peroxide-catalase (0.1 mg/tablet) systems were also tested. Fungal isolates suspended in hydrogen peroxide (3%) at a concentration of 10⁶ CFU/mL were placed into the containers provided by the manufacturers, which were filled up to the recommended level. The enzyme catalyst was present during the incubation as instructed by the manufacturers. Arion Cronos and Oxysept Comfort were tested after the completion of the neutralization process (1 and 6 h, respectively). A sterile phosphate-buffered saline solution was also inoculated with fungal conidial suspensions and served as a positive control. Aliquots (5 μL) of suspension were removed for analysis after 1 h (Arion Cronos only), 6, 8, and 24 h and inoculated onto Sabouraud dextrose agar. The culture plates were incubated at an appropriate temperature and readings were taken when growth appeared on the positive control plate. The results were recorded as growth positive or growth negative. Extreme care was taken to avoid aerial contamination and cross contamination. The assay was performed in duplicate. In this study, the primary endpoint was no fungal growth after exposure to the disinfecting solution.

Results

Manual counting of fungal colonies on agar plates confirmed that conidial suspensions prepared to an optical density equal to 0.5 McFarland standard had a concentration of ∼10⁸ CFU/mL.

Results of susceptibility testing are reported in Table 2. Both assays yielded identical results. The fungal species recovered from the suspension tubes after incubation were confirmed by growth characteristics to be identical to those used for inoculation.

The best results were given by 3% hydrogen peroxide-based Arion Cronos and Oxysept Comfort solutions, which were able to kill most of the fungal isolates within the minimum recommended disinfection time (1 and 6 h, respectively). However, only Oxysept Comfort without catalase was able to eradicate all the fungi tested after a 6-h exposure. All the other disinfecting solutions were partly ineffective, killing only some of the fungal isolates, even after a 24-h exposure. The worst results were exhibited by Regard and Dua Elite solutions, which, after a 6-h exposure (minimum recommended disinfection time), showed growth of 12 and 10, respectively, of the 13 fungal strains tested.

Overall, based on the ratio “number of growth-negative cultures/total number of cultures,” after a 6-h exposure the susceptibility of the fungal species to the disinfection solutions tested was, in the descending order, Fusarium spp. (16/21)>A. fumigatus (15/21)>A. flavus (18/42)>Curvularia sp. (2/7). In our experiment, only Oxysept Comfort was effective against the single isolate of Curvularia sp.

Discussion

Fungal keratitis is potentially blinding and often misdiagnosed among contact lens wearers. In developed countries, the incidence of fungal keratitis is generally low and infection among contact lens wearers is much rarer when compared with bacterial and Acanthamoeba keratitis. The treatment of fungal keratitis is challenging and the relative risk of penetrating keratoplasty is estimated to be higher in fungal keratitis compared with bacterial keratitis. ¹¹ Despite intensive medical therapy, between 11% and 36% of patients with fungal keratitis can still be expected to require a corneal transplant at some stage of their infection (usually after the acute infectious event) for the treatment and visual rehabilitation of a quiescent, noninfective corneal scar. ⁵

Fungal keratitis has been reported worldwide, but it is more common in tropical and subtropical regions. In developing countries, ocular trauma is the main predisposing factor for fungal keratitis.^11,12 In the United States, the reported incidence of fungal keratitis is 30,000 new cases per year. ⁵ Historically, ocular trauma with vegetable matter or objects contaminated with soil was the major risk factor for fungal keratitis in the United States, ¹³ whereas fungi accounted for less than 5% of all episodes of contact lens-related microbial keratitis.^1–4 However, this pattern appears to be changing, with the number of patients with contact lens-associated fungal keratitis becoming equal to or even exceeding the number of patients with trauma-related fungal keratitis.^5,14,15 Although the recent outbreaks of fungal keratitis associated with contact lens use have involved mainly the Fusarium species,^9,10 Aspergillus spp. are also very common causes of keratomycosis in many parts of the United States and the Asia-Pacific region. ¹³

Contact lens wear is a major risk factor for the development of microbial keratitis. ¹⁶ Studies of patients with contact lens-associated corneal ulcers have shown microbial contamination of the care systems, which may represent the source of the infecting organisms.^17,18 Contamination rates range from 20% to 80% in asymptomatic and symptomatic patients, with Pseudomonas aeruginosa emerging as the predominant pathogen in the presence of other bacteria, fungi, and Acanthamoeba.^16,19,20 The presence of microorganisms in the contact lens cases does not necessarily always imply the occurrence of keratitis. Various factors could explain the discrepancy between case contamination rates and the occurrence of contact lens-related keratitis. On the one hand, in most cases, the host reaction could overcome a microbial infection in its initial phase and prevent the development of true keratitis. On the other hand, some organisms may be poorly adherent to the corneal epithelial cells and, therefore, not capable of colonizing the ocular surface. ²¹

In general, proper contact lens disinfection is crucial in preventing contact lens-related microbial keratitis. Nevertheless, despite the apparent adherence to recommended disinfecting regimes, several studies have shown a significant degree of microbial contamination of the contact lens cases.^16,19 Biofilm formation on the internal surfaces of the contact lens case may be responsible for disinfectant failure by providing a continuous seed inoculum.^19,22 Furthermore, experimental evidence suggests that currently available contact lens disinfecting solutions have differing levels of efficacy on different microbial organisms.^23–25 Overall, solutions are more effective against bacteria than fungal organisms or Acanthamoeba.

Soft contact lens solutions have evolved over the years. Current products cause fewer toxic and hypersensitivity reactions and are easier to use than previous products, but they are less effective in killing organisms, especially those producing resistant forms. For example, multipurpose solutions containing PHMB and polyquaternium-1 (Polyquad) were found to be ineffective against Acanthamoeba cysts and Bacillus cereus spores.^6,23 Hydrogen peroxide 3%, one of the oldest disinfection solutions for contact lenses, has a respectable antimicrobial activity, but is toxic to human tissue as well. Therefore, it is necessary to neutralize fully any hydrogen peroxide adherent to the lens before the lens is reapplied to the eye. This can be done by enzymatic means (catalase) in a 1- or 2-step process. In the 1-step system, a catalase tablet is added to the lens case at the beginning of disinfection. This system, generally active against bacteria and fungi, proved to be ineffective at killing B. cereus and Acanthamoeba within the minimum recommended disinfection time.^6,23 Because the enzyme catalyst is present from the very beginning of the disinfection step, the hydrogen peroxide is neutralized long before complete disinfection can occur. Conversely, complete disinfection may be accomplished by using the 2-step system, with neutralization occurring after a 9-h exposure (overnight) to hydrogen peroxide.

Relatively little attention has been paid to the efficacy of disinfecting solutions against fungi.^7,22,26–33 In our study, we investigated the susceptibility of 13 filamentous fungal strains isolated from human corneal ulcers to 6 contact lens disinfecting solutions. On the whole, 3% hydrogen peroxide-based Arion Cronos and Oxysept Comfort demonstrated a good fungicidal activity; indeed, they were able to kill most of the fungal isolates within the minimum recommended disinfection time (1 and 6 h, respectively). However, only Oxysept Comfort without catalase was able to eradicate all the fungi tested after a 6-h exposure. All the other disinfecting solutions were partly ineffective at killing some of the fungal isolates within the minimum recommended time and, in some cases, even after a 24-h exposure. The worst results were given by Dua Elite, containing PHMB 0.0001% and sodium hyaluronate, and Regard, containing the chlorite/peroxide complex.

As for Complete RevitaLens, containing polyquaternium-1 and alexidine, it is interesting to note that all 3 isolates of Fusarium spp. were eliminated after a 6-h incubation. This finding may seem contrary to the observations made during the worldwide Fusarium outbreak, which was linked to the use of alexidine-based ReNu with MoistureLoc.^8–10 Actually, this is not the case, because Complete RevitaLens, apart from alexidine, which may be ineffective at eradicating species of Fusarium, also contains polyquaternium-1, which has been shown to be effective against this organism.^22,29

Our experiment also showed that different corneal fungal isolates belonging to the same fungal species may show different susceptibilities to the same disinfecting solution. This must be taken into consideration while testing the antifungal activity of new disinfecting solutions.

In our assay, like in other published studies, the fungal inoculum size was 10⁶ CFU/mL.^{7,28,29,32,33} The exact relevance of this concentration in the contamination of contact lens care systems remains to be established, because the size of fungal inoculum causing contact lens-related keratitis is not known. Under the experimental conditions used in this study, 3% hydrogen peroxide without the neutralizer showed the highest antifungal activity. Nevertheless, it is still possible that the other disinfecting solutions may have a sufficient activity, if lower fungal concentrations are tested.

Even though we analyzed a significant number of ocular fungal isolates, a clear limitation of our study is that we used suspended organisms rather than biofilm, which would be more resistant to killing.^22,30 In fact, most contact lens cases develop biofilm with use. Another limitation is that we tested only one isolate of Curvularia sp.; therefore, no definitive conclusion can be drawn about this fungal species. Last, but not least, we did not assess any environmental or other clinical nonocular filamentous fungal isolates.

In the United States, only 1-log reduction of fungal organisms within the recommended disinfection time is required by the Food and Drug Administration (FDA) to meet the primary Stand Alone Test criteria for contact lens disinfection. However, the recent appearance of reports showing an insufficient antifungal activity of multipurpose solutions is of great concern. Indeed, Ide et al. ²⁸ found that the antifungal activity of contaminated multipurpose solutions may be insufficient to prevent fungal colonization of contact lens materials despite meeting or exceeding the FDA Stand Alone Test criteria. In another article, Boost et al. ²⁹ reported the inability of 3 multipurpose solutions to achieve a 90% reduction against fungal isolates and recommended extension of the current FDA testing panel for multipurpose solutions to include additional fungal isolates.

On the whole, our data, like the results from other studies, suggest that most contact lens disinfecting solutions may not be effective enough, if contact lens care systems become contaminated with filamentous fungi. As a result, the need for a complete re-evaluation of the real antifungal efficacy of currently available contact lens disinfecting solutions must be stressed. This is crucial, to reduce the risk of fungal keratitis in contact lens wearers, especially in tropical and subtropical regions, where keratomycoses are common.