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An Overview of the Challenges to the Development of a Consensus Methodology of Disinfection Efficacy Testing for Contact Lens Care Products

Brocious, Jeffrey, M.S.; Tarver, Michelle E., M.D., Ph.D.; Hampton, Denise, Ph.D.; Eydelman, Malvina, M.D.

doi: 10.1097/ICL.0000000000000470
Review Article

Abstract: With the increasing incidence of more pathogens that can cause microbial keratitis (MK), it is necessary to periodically reassess disinfection multipurpose solutions testing requirements to ensure that relevant organisms to challenge them are being used. Current testing protocols have included common pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus, Serratia marcescens, Candida albicans, and Fusarium solani but have omitted less common pathogens such as Acanthamoeba. Specifically, Acanthamoeba sp. has recently been identified as a prevalent cause of MK in certain countries. Developing an appropriate protocol for this unique organism presents a challenge, given its two distinct life stages, methods to grow the organism, encystment techniques, and many other parameters that can affect testing outcomes. Therefore, the appropriate combination of these parameters is crucial to developing a protocol that ensures consistent, accurate results. The FDA has recognized the importance of establishing a standardized testing protocol for this pathogen and embarked on research efforts to provide a recommended testing protocol for testing contact lens care products.

Office of In Vitro Diagnostics and Radiological Health (J.B.), Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD; and Office of Device Evaluation (M.E.T., D.H., M.E.), Center for Devices and Radiological Health, Food and Drug Administration, Silver Spring, MD.

Address correspondence to Malvina Eydelman, M.D., Office of Device Evaluation, Center for Devices and Radiological Health, Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, MD 20993; e-mail:

The authors have no funding or conflicts of interest to disclose.

Accepted December 04, 2017

With an estimated 40.9 million contact lens users in the United States,1 adequate cleaning and disinfection of contact lenses with contact lens solutions has significant public health impact. Currently, the International Organization for Standardization (ISO) document 147292 and the FDA care product guidance3 describe protocols (stand-alone and regimen tests) for assessing the disinfection efficacy of care product solutions against a variety of organisms (Staphylococcus aureus, Pseudomonas aeruginosa, Serratia marcescens, Candida albicans, and Fusarium solani). Unlike bacteria and fungi, amoebae have not been included in the disinfection efficacy testing recommendations. At the time of publication, ISO 14729 recommended against including Acanthamoeba as a challenge organism based on its rare incidence of infection, preventable source of contamination, and lack of standardized testing methodology. However, within the last two decades, the number of reported Acanthamoeba infections has increased among contact lens users.4

Acanthamoeba keratitis (AK) is a rare, but sight-threatening corneal infection presumed to be caused by the trophozoite stage of the Acanthamoeba protozoan. Many species of the organism, including Acanthamoeba castellanii, A. polyphaga, A. hatchetti, A. culbertsoni, A. rhysodes, A. lugdunensis, A. quina, and A. griffin, can cause significant keratitis among contact lens users.5 In 2007, cultures from 158 patients that tested positive for AK between March 16, 2007, and July 10, 2007, resulted in the voluntary recall of the multipurpose solution (MPS), Complete Moisture Plus, suspected to be associated with this outbreak.6 Patient factors associated with this outbreak included the topping off (adding additional solution to the case vs. solution replacement) of solutions in the contact lens case and wearing contact lenses for 5 years or less. It has also been demonstrated that this solution was able to induce encystment after exposure within 6 to 24 hr of incubation, which may have also contributed to this outbreak.7 After the recall, however, the number of AK cases in the United States did not decrease to their preoutbreak levels.4 Some researchers have cited poor contact lens hygiene, failure of MPS to disinfect against amoebae, increased awareness of the condition, and better diagnostic tests (e.g., confocal microscopy) as contributing factors to the persistence of AK cases.

Acanthamoeba species, which exist in nature in two different forms, trophozoite and cyst stages, have caused infectious keratitis.5,6 It is presumed that the trophozoite stage of Acanthamoeba causes infection by binding to mannose glycoproteins within a compromised corneal epithelium and secreting cytolytic proteins and proteases that promote further penetration.8–12 The genus nomenclature is derived from the Greek “acanth,” meaning “spikes,” because of the acanthapodia found on the exterior of the cell membrane. The organism is found ubiquitously in the environment, mainly in soil and in environmental sources of water, including tap water. In addition to species categorization, the organism is also classified based on its genotype with each having distinct morphologic characteristics. Currently, there are at least 18 genotypes, with the most common clinical isolate being genotype T4.13 Acanthamoeba is capable of encysting during periods of environmental adversity. After either a chemical or physical stimulus, the trophozoite will produce two distinct walls, the endocyst or “inner wall” and ectocyst or “outer wall.”13,14 They are then able to withstand extreme conditions including high temperature, high salinity, chlorine, and over 20 years of desiccation.15

Despite many public health initiatives undertaken by national and international standard bodies, manufacturers, healthcare providers, and regulatory agencies to aid in mitigating ocular infections, some users still develop corneal infections in part because of ineffective solutions. Testing solutions can be quite challenging because a protocol for disinfection cannot account for every infectious organism or every potential scenario of patient misuse. For example, the protocol to challenge solutions against bacteria is not applicable to amoebae, given that they are two distinct, metabolically different organisms. Although the current panel of organisms recommended in ISO 14729 and FDA guidance were deemed representative of most keratitis infections at the time these documents were drafted, and protocols for testing additional organisms may be helpful to assess the activity of relatively newer infections such as Acanthamoeba as recommended by FDA's 2008 Ophthalmic Devices panel meeting.16

Although the importance of a consensus disinfection testing protocol against Acanthamoeba is discussed in ISO 14729, current standards and FDA guidance documents do not include an assessment of efficacy against the organism. To address the potential for solutions to induce encystment that may have played a role in the Acanthamoeba outbreak in 2007, ISO 19045 was published to provide a method to evaluate this possible outcome. However, as this information is important, we still need to address the biocidal effect of solutions.17 Because its inception as a significant ocular pathogen, protocols to test solution efficacy against this organism have been widely debated primarily because growth characteristics of amoebae are significantly different than those of bacteria and fungi. As such, a protocol that renders accurate and repeatable results continues to remain a challenge.

Testing of contact lens solutions for efficacy against Acanthamoeba before introduction into the US market was first recommended at a 2008 meeting of the Ophthalmic Advisory Panel.16 Subsequently, the 2009 FDA workshop fostered a discussion on possible microbiological approaches for disinfection efficacy testing against Acanthamoeba.18 The strain type, life-cycle stages, growth method, and encystment techniques were all mentioned as important factors in the development of a disinfection efficacy testing protocol. After the workshop, a great deal of research was conducted to further develop a protocol to test for efficacy against this pathogen. Many investigators' research described various methods for each component of a suitable protocol. The species of Acanthamoeba, type of strain, method of trophozoite and cyst production, inoculum preparation, inoculum size, neutralization methods, and enumeration of the organisms after exposure to the solution were all investigated; however, agreement regarding a single protocol has not yet been reached. The FDA began to address these challenges by comparing growth methods, encystment methods, and different strains of Acanthamoeba. The outcomes of this research demonstrated that differences in methods produce variable results, which further bolstered the need for a standardized protocol.19

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Choosing an appropriate Acanthamoeba species and strain plays an important role when deciding which representative test organism(s) to challenge a solution. Like bacteria and fungi, different species of Acanthamoeba may exhibit different susceptibilities to a single solution. In addition, it is important to select an organism that represents most keratitis infections. Previous studies have performed testing of solutions using diverse species and strains of the organism; however, it is not practical to test all strains that have been known to cause infection. Historically, A. castellani is the species used most often in protocol development studies because of its prevalence in clinical infection. Strains of A. castellani that have been tested are 1501/A from the Culture Collection of Algae and Protozoa (CCAP),20 30234 from the American-Type Culture Collection (ATCC),21 ATCC 30868,22,23 ATCC 50370,19,24–27 ATCC 50514,24,26 ATCC 30234,21,28 Centers for Disease Control (CDC) strains,19 and various clinical and environmental isolates.29,30 In addition, many strains of A. polyphaga such as ATCC strains 3087331 and 30461,21,25 Ros22,23,27 and CCAP 1501/3g27 strains have been used to challenge solution efficacy. The most appropriate species and strain(s) for testing would likely be those associated with clinical infection and resistance to disinfection.

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The method for trophozoite and cyst stage propagation also has not been standardized for an efficacy protocol. It is important to consider the growth method because it may have an impact on the test system as well. For example, some studies grow trophozoites using proteose peptone glucose broth or peptone-yeast extract glucose medium, which provides nutrients to the organism (axenic growth) in the place of their natural food source or bacteria.20,21 Other studies use nutrient broth spiked with antibiotics such as penicillin or streptomycin, presumably to prevent bacterial contamination,22,25,28 whereas other studies have used trophozoites that have been grown using bacteria as their food source (bacterized growth).19,29,30 These influences on growth conditions may or may not render the organism more susceptible to solution testing, which could ultimately yield inaccurate results. In addition, results from different methods may not be comparable because of these influences, hence the need for standardization of the methods.

In addition to trophozoite testing, it is important to challenge solutions with preparations of cysts. After exposure to the right stimulus, a cyst can revert back into its trophozoite form (the presumed infective stage); therefore, it may be important to consider this conversion when evaluating disinfection efficacy. For example, a solution that is effective against the trophozoite form may not be effective against its cyst form. As a result, the cyst may sustain disinfection, excyst after exposure to the ocular environment, and potentially cause infection. It has been demonstrated that encystment techniques also may impact the outcomes of testing. When designing a robust protocol for disinfection, an encystment technique that yields the hardiest cysts is desirable because that represents a worst-case scenario and, therefore, represents more of a challenge. Some investigators allow trophozoites grown axenically to naturally encyst on agar for 2 weeks and20,28 others encyst by exposing trophozoites to bacteria first and then incubating for several weeks.19,21,31 Still others use different media such as Neff's constant pH encystment medium24–27,31,32 or media spiked with magnesium chloride22,23 to chemically induce encystment or prepared by incubating trophozoites in phosphate-buffered saline plus heat-killed yeast cells.28 Similar to trophozoite production, different methods may have an impact on cyst susceptibilities to contact lens solutions. For example, Shoff demonstrated that the starvation method yielded a lower log kill of cysts than using Neff's medium to encyst.19 Therefore, it may be helpful to consider implementation of one encystment method to avoid inconsistent results with respect to differences in cyst susceptibility.

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Similarly, starting concentrations for each trophozoite and cyst inoculum vary significantly among studies. The starting concentrations from investigators have ranged between 50 organisms/mL30,32 and 250,000 organisms/mL.27 Comparing results from highly variable-starting concentrations may not be appropriate because log kills are not necessarily comparable at significant differences in concentration. For example, a 2-log kill from a starting concentration of 100 organisms/mL (or 99 organisms killed) is significantly different than a 2-log kill of a 1,000,000-organism/mL starting concentration (or 990,000 killed organisms). Therefore, it is important to select an appropriate range to be able to achieve countable decreases in log kill while still narrow enough to minimize variability in results.

Testing solutions at various time points of exposure to Acanthamoeba requires the use of a neutralization step to ensure that the solution has ceased its biocidal activity against the challenge inoculum at that specified time point. Most studies include this additional control and have used solutions such as Dey–Engley broth,19,20,26,28–30 bovine liver catalase,31 or solutions that contain different concentrations of Tween 8022,25,31 or combinations of lecithin and polysorbate23 to neutralize their solutions. In addition, it may be acceptable to dilute the solution in enough diluent (e.g., Page's Amoeba Saline) to reduce the biocide concentration to render it nonbiocidal. When conducting tests that involve neutralizers, it is important to consider conducting control studies to demonstrate that the neutralizer has negated the effect of the biocide. For example, ISO 14729 describes a recovery medium control test to ensure that bacteria/fungi can be recovered from neutralized solution.2 These studies may consist of adding a small inoculum to a “neutralized” solution and determining viability after incubation.

To add further complexity, the final enumeration of Acanthamoeba after exposure to the solution varies in several studies. For example, some investigators use bacterial plaques coupled with the Reed and Muench computation method in determining viability.31 Others use the Spearman–Karber computation method22,23,25–27 by culturing Acanthamoeba in microtiter plates or use the Beattie et al. computation method19,20 by growing them on agar plates with bacteria. Other methods describe the use of microscopic observation and even flow cytometry to determine viability.21,24 The sensitivities of these methods can vary significantly; therefore, it is important to choose a method that is not only optimal for the recovery of viable organisms but is also coupled to a robust and accurate computation method.

Given the recommendations made at the 2009 FDA workshop and the vast amount of subsequent research to further specify best methods, another microbiology workshop was cosponsored by FDA, the American Academy of Ophthalmology (AAO), the American Academy of Optometry (AAOpt), the American Optometric Association (AOA), and the Contact Lens Association of Ophthalmologists, Inc. (CLAO) in 2014.33 One of the goals of the workshop was to determine uniform testing methods for Acanthamoeba disinfection efficacy. Workshop panelists recommended that ATCC strains 50370 (A. castellanii) and 30461 (A. polyphaga) be used because these strains represented commonly isolated species from clinical specimens that contributed to keratitis.34 In addition, the panel recommended that a disinfection efficacy method includes axenic growth for trophozoites and the starvation method for encystment.

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A well-designed protocol assesses the efficacy of solutions by determining the relative number of live microbes eliminated (i.e., log kill) after exposure to the contact lens solution for the manufacturer's recommended disinfection time. For a solution to be labeled effective, it must meet predetermined established acceptance criteria. The FDA Care Product Guidance3 and ISO 147292 both state that the primary criteria of the stand-alone protocol require a 3-log kill for each species of bacteria and 1-log kill for each species of fungi for a solution to be labeled for disinfection. If a contact lens solution fails these primary criteria, it must pass the secondary criteria of the protocol, in which a cumulative 5-log kill for all bacteria and stasis for both fungi must be obtained. If a solution passes the secondary criteria of the stand-alone test, it must then pass the regimen test, which incorporates the labeled disinfection regimen of that solution and may be composed of cleaning, rinsing, and soaking steps. The acceptance criteria of the regimen test are more stringent than the stand-alone criteria in that the solution must result in at least a 4-log kill or 99.99% reduction in concentration for each organism tested. If the solution passes the regimen test criteria, it would be appropriate to be labeled as part of a disinfection regimen. However, it is not clear what the appropriate criteria should be with respect to acceptance criteria for log kill of Acanthamoeba. Panelists from the 2014 workshop noted that a 2- or 3-log kill for trophozoites was adequate for disinfection, but that more research was needed to establish a criterion for cyst kill.34 Variability among each method was identified as a critical concern, given that differing methods may lead to different outcomes.

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Over the past decade, there has been a multitude of studies that have attempted to validate a protocol to determine disinfection efficacy against Acanthamoeba. Selection of species/strain, growth method, encystment technique, suspension media, concentration, neutralization, and enumeration may have an impact on disinfection efficacy results. Therefore, appropriateness of each method within each parameter is important to be assessed. Given the extreme diversity of the methods used to determine contact lens solution disinfection efficacy against Acanthamoeba, a standardized protocol that has been tested across laboratories and addresses the worse-case scenarios (e.g., most resistant cyst) may be helpful to demonstrate accuracy and reproducibility. In addition, a standardized protocol would provide a framework to better evaluate and select appropriate acceptance criteria for disinfection. In an effort to address this outstanding issue, FDA sought to further refine its basic protocol initially developed by Shoff et al.19 by incorporating recommendations (e.g., test strains and growth methods) from both FDA workshops. These refinements and results from a study conducted in collaboration with the Winchester Engineering Analytical Center (WEAC) are further discussed in a publication by Fedorko et al. entitled, “Optimized Protocol for Testing Multipurpose Contact Lens Solution Efficacy Against Acanthamoeba.” The publication describes this modified protocol and results from an evaluation of interlaboratory and intralaboratory reproducibility of axenically grown trophozoites and cysts that were challenged against a variety of solutions with different preservatives. Results indicated that the protocol was able to effectively evaluate Acanthamoeba efficacy testing by demonstrating reproducibility. The publication can be viewed in this edition of Eye and Contact Lens.

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Contact lens solution; Acanthamoeba; Contact lens disinfection testing; Regulatory science

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