Experts support development of efficacy standards for Acanthamoeba testing

Most eye care providers still remember the 2007 Acanthamoeba keratitis outbreak in contact lens wearers.

Concern has grown among practitioners over the ability to test for this organism in contact lens products, but there remains no universally accepted method for testing disinfection efficacy against such microbes, according to a press release from the American Academy of Ophthalmology.

In September, the U.S. Food and Drug Administration Center for Devices and Radiological Health, the American Academy of Optometry, the American Academy of Ophthalmology, the American Optometric Association and the Contact Lens Association of Ophthalmologists hosted a workshop to develop new approaches to standards for microbiological testing methods.

The November issue of Primary Care Optometry News presented the participants’ discussion on emerging pathogens in keratitis and the role of soil in disinfection efficacy testing (“Experts agree on need to standardize testing for contact lens products”). This second part of the two-part special report will address disinfection efficacy test methods for Acanthamoeba.

Acanthamoeba: Organism test methods

Currently, ISO standard 14729 does not recommend acanthamoebicidal testing, Daniel P. Fedorko, PhD, said in his opening presentation.

He detailed the variables in published test methods, which include the trophozoite production, cyst production, inoculum preparation, challenge inoculum size, neutralization of disinfecting agent, enumeration of surviving organisms after challenge and the test organisms themselves. There are various test organisms from both the American Type Culture Collection (ATCC) and the U.S. Centers for Disease Control and Prevention – A. castellanii, A. polyphaga, A. hatchetti, A. royreba – as well as clinical isolates.

Fedorko detailed the FDA proposed protocol for Acanthamoeba testing, which includes the use of A. castellanii and bacterized trophozoite production. He acknowledged, however, that there is no universally accepted protocol, and that published methods vary widely in critical components.

Fedorko encouraged the panelists to move toward the standardization of efficacy testing for Acanthamoeba.

Moderator Deepinder K. Dhaliwal, MD, LAc, prompted the panelists to consider whether ATCC 50370 A. castellanii is the most suitable strain for testing.

Many of the panelists explained that it was an easy strain to obtain and work with, thus supporting it as an appropriate choice.

“It’s available from the ATCC,” Simon Kilvington, PhD, said. “It’s relatively easy to grow in the laboratory and in our hands it makes a good cyst for testing.”

“The castellanii is certainly a good option,” Ralph Stone, PhD, said. “Polyphaga would be a good option as well. Both are T4 and they are interesting.”

Elmer Y. Tu, MD, however, disagreed with his fellow panelists, arguing that the strain is outdated and not representative of the strain currently seen in patients.

“My earlier presentation definitively showed that all current solution systems are inadequate to prevent significant levels of Acanthamoeba infection clinically,” he told Primary Care Optometry News after the workshop. “All solutions are likely currently associated with rates of infection that exceed the outbreak of Acanthamoeba keratitis of the late 1980s.”

He said in the panel discussion: “Having an isolate from 1978 from a human keratitis specimen gives me pause, especially with the potential that we’re dealing with new strains and virulence over the last several years. It may not reflect, in any way, some of the virulence factors that we’re seeing in the most recent outbreak.

“I know that the FDA has worked with the CDC and V568, V572 and V573 and has done some preliminary testing on that,” Tu continued. “I’m sure we could ask the CDC to make those available, and they would be strains from the most recent outbreak.”

The other panelists reviewed the rationale for the current strain and shared some suggestions for adding new strains.

“The strain 50370 is in there because we need to get consensus and standardization in testing,” Kilvington stated. “We should all be able to take 50370, grow it up in the agreed manner, produce cysts from it and all get the same results. Only when we start doing that should we begin thinking about current strains. And only through testing will we be able to show that these new strains are, in fact, more resistant.”

“When we start looking at new strains, we have to remember that we have to get them into the ATCC culture system in order to maintain a realistic understanding of how that organism is going to perform long term,” Stone said.

Donald Ahearn, PhD, also recommended that the strain be sequenced so that researchers could better understand its genotype.

Axenic vs. bacterized

Panelists then discussed the advantages and disadvantages of using the axenic or bacterized growth method for trophozoites.

Some of the panelists stated their preference of using the axenic method, citing numerous disadvantages for the bacterized method.

“My problem with bacterized cultures would be the fact that Acanthamoeba may express a selective genome via lateral gene transfer,” Ahearn said. “They have a selective feeding procedure. So if grown in mixed cultures, they’ll sort out the food and reflect that in their proteome.

“Bacterized trophozoites may not provide a true expression of their capabilities in an environment such as a shower drain,” he continued.

Kilvington concurred: “You can argue that bacterized is more real world, but it’s not, because the real world isn’t a store case. And, from my experience, axenic are much easier to control and to get consistency with. We have done comparisons between axenic and bacterized cultures for trophozoites and found that there isn’t much difference between them.”

“The axenic culture is preferred for a standard method,” Mary Mowrey-McKee, PhD, said. “Bacterized have confounding results because you’re killing the bacteria at the same time you’re trying to kill the trophozoites or the cysts.”

Tu disagreed, indicating that the bacterized method better reflects real-world scenarios.

“An FDA paper from a couple of years ago demonstrated that bacterized were more resistant to the multipurpose solutions, or contact lens disinfection solutions, than the axenically grown trophozoites,” he said. “And as Dr. Fleiszig mentioned earlier, if you keep cycling them as axenic organisms, which they are not, that’s not how they exist in nature, and they are going to have different characteristics than in a contact lens case or in a contact lens wearer.

“You have to be careful about extrapolating, and we’re already on thin ice here trying to extrapolate a standardized testing method for Acanthamoeba when we don’t have necessarily good follow-up on whether it’s going to work or not,” he continued. “And so we have to guess, and my guess would be bacterized.”

Stone expressed additional concerns about the use of bacterized systems.

“One of the key things is that I’m going to have a mixed culture at the end,” he said. “I’m going to have Acanthamoeba and I’m going to have one of the bacteria, so I’m going to ask the disinfectant to work on both. If these were nice, linear systems, it might be possible, but they are not.”

“I think disinfection is nonlinear,” Tu countered. “In a real world situation, it’s not linear. We’re now looking for solutions that aren’t necessarily going to be killing Acanthamoeba in a contact lens case. That’s never going to be the situation you are dealing with.”

In terms of encystment method, all panelists agreed upon the use of the starvation method.

“Starvation would be the best way,” Mowrey-McKee said. “This is the most real-world situation for the way the cysts are produced in nature and so, with the chemical methods, you may alter the quality, the characteristics, of the cysts compared to the way they would grow in the wild.”

Appropriateness of one log kill

Panelists then addressed if a one log kill for trophozoites and stasis for cysts was an appropriate amount of killing in terms of testing solutions working against Acanthamoeba.

Many, like Tu, felt that a three log kill was both necessary and achievable for trophozoites, and that stasis for cysts was not enough.

“The question, I think, before the panel is what are we trying to achieve by testing, and perhaps increasing, the efficacy of solutions against Acanthamoeba?” Tu asked. “Is it to maintain the status quo? Or is it to try to return us closer to what our pre-outbreak incidence is? I think for most people it would be to reduce, or try to get it back to what it was before. And that is the answer to your question. If the current solutions already achieve a one log kill of trophozoites, then it’s not sufficient.”

Kilvington expanded: “Before you can start talking about acceptance criteria, you need to have a standardized method of determining the susceptibility of trophozoites and cysts to these products. As I showed in my findings, modern contact lens solutions, those based on dual biocidal agents, are pretty effective against trophozoites, and you would exceed one log kill.

“You’re always going to struggle to kill the cysts and you just have to accept that most contact lens care systems cannot deal with Acanthamoeba cysts,” he continued. “Stasis is not acceptable because we are saying then it’s fine for the cysts to be alive but not existing in contact lens solution. If they’re alive and viable, they will hatch out and cause infection.”

Ahearn and Mowrey-McKee both expressed caution in utilizing stronger solutions.

“You have to be careful here,” Ahearn stated. “If you increase the strength of the solutions, you may facilitate differentiation to cysts. Cysts in bathroom environments are induced by shocks from temperatures and various types of disinfectants.”

“We have to be careful that we’re not going to damage the cornea,” Mowrey-McKee said. “If you increase the concentrations of the microbial agent, you’re going to most likely cause damage to the corneal epithelium and then make it even more susceptible to infection because you’ve already damaged it.”

Kilvington responded that modern solutions based on dual biocides have displayed efficacy and not compromised the cornea. However, he worried about products that would not meet a three log kill standard.

“What will you do about the solutions on the market today that give you less than one log kill?” he asked. “They’re used quite successfully, it seems. They are not being implicated in outbreaks of Acanthamoeba or other forms of keratitis, but what will you do about solutions if you start setting acceptance criteria that are currently on the market and would not meet these standards?”

Mowrey-McKee also commented on biocidal uptake by lenses.

“Manal Gabriel has data showing that biocidal uptake with different lenses, with different solutions, does affect efficacy,” she said. “At the Contact Lens Institute, we developed a method which is now in ISO and it will become a standard at the end of this year, 18259. It is measuring the disinfecting efficacy of a solution in the lens case, with various contact lenses, with a panel of organisms in organic soil – the panel that is in 14729.”

Panelists next addressed whether the same killing criteria would be acceptable in the presence of a contact lens.

“If you put in a certain contact lens and allow it to deplete the biocide, then you’ve lost that for its activity against Acanthamoeba,” Kilvington said. “Our test methods are not designed to be real world. We’re only interested in reproducibility at this stage. If you want to ramp things up, you would certainly be in the situation where you’re getting a very good kill in a test tube. But when you repeat the experiment in the presence of certain contact lenses, you’ll find that the activity is significantly reduced.”

Stone agreed: “These models can only go so far. I cannot control the environmental portion of this. We’ve got to be very careful that we don’t put standards in place now that are so high that none of us can live with them.”

Patient education, water use

Mowrey-McKee encouraged a commitment to educating patients in how they handle their lenses, which can help in the fight against infection.

“There needs to be a big educational effort so that patients don’t abuse their lenses or use water,” she said. “They have to know that water is a huge risk factor for keratitis, and I would assume from ocular infections that people are not aware of that.”

Kilvington and Tu also addressed the role of water in ocular infection.

“You won’t find Acanthamoeba in tap water every time you look because it’s sporadic,” Kilvington said. “The epidemiology and the hard facts are quite clear that contact lens wear and tap water don’t go together, as with contact lens wear and showering, and contact lens wear and swimming. They are high-risk practices. That goes for gas-permeable lens wearers as well.”

Tu said small geographic variations have been shown even within a city.

“To do a study, you would need to go into homes and not only measure Acanthamoeba, but also disinfectant levels and everything else,” he said. “It’s far from conclusive that the U.S. outbreak has nothing to do with tap water. I think the exact opposite.

“The overarching theme here is that the multipurpose solutions were very good and they were very well-accepted,” he continued. “But things change, and we’re faced with a different risk now than 10 years ago.”

Tu also noted that even patients who use daily disposable lenses have a risk of Acanthamoeba.

“The study that John did in the U.K. with daily disposables had a couple of Acanthamoeba infections,” he stated. “It’s not entirely the solutions. You are looking at a higher environmental risk at this point.”

Malvina Eydelman, MD, discussed how educational efforts could be improved, remarking that the FDA has made a large effort in educating patients and doctors and would like other organizations to contribute parallel efforts.

“We don’t regulate doctors and patients,” she added. “What we do regulate is devices. And as such we can have premarket requirements of certain safety standards prior to getting these devices on the market. I agree in that whatever we do in the realm of education needs to be done in parallel with continuing to try to strengthen our premarketing activities. And that’s what the goal of today’s workshop is.”

The panel members were asked if they believe that gas-permeable lens wearers would stop rinsing their lenses in tap water even with educational campaigns and labeling changes.

“History tells us that it’s difficult to change behaviors,” Tu said. “If it’s an effective campaign you might be able to improve patients’ behavior. It’s worthwhile because education is important. All of us, as eye care professionals, have suffered from reduced compensation and reimbursement for actually spending time with our patients to talk to them about lens care, and I think that’s something they need on a one-on-one basis, which, historically, has been the most effective.”

Dhaliwal recounted a specific situation with one of her patients who developed Acanthamoeba after using water with her GP contact lenses and was not aware of the risk.

“When I told her that rinsing in tap water is definitely a problem with contact lens wear, she said, ‘You know, I wish someone had told me that. If someone had told me that, I would have done whatever they recommended,’” she said. “When patients know that blinding infections can result, they change their behavior.”

Loretta Szczotka-Flynn, OD, PhD, acknowledged the importance of providing patients with the tools necessary to change their behaviors along with education.

“As a clinician, I tell my patients not to use tap water to rinse their gas-permeable lenses. However, a majority of them say, ‘Well, I’ve been doing it for 30 years. Why should I change now?’ And the second thing is, ‘What do I rinse it with?’ she said.

The attendees debated about which products should be used to provide copious rinses of harsh cleaners from GP lenses and which, if any, current products on the market are approved for GP lenses, but there was no consensus on these products’ efficacy or safety.

Eydelman encouraged the present organizations to speak with manufacturers about delivering an alternative solution.

James Cook, of Abbott Medical Optics, said that a manufacturer needs to be assured of a promising marketplace.

“If contact lens organizations [foster] that market through education, it would follow that we could develop these products,” he said.

Conclusion

Several of the panelists expressed the desire to continue the discussion among themselves and their organizations to completely standardize testing protocols and improve the health and safety of their patients.

“The most important topic that keeps being reiterated in the summary remarks is that we’re not done,” Eydelman noted. “Today should be viewed as just the beginning of the next stage. We have made enormous progress, but I want to urge you to look at this as a springboard. There are some really good, strong recommendations that came out from each of the panels, and we’re going to be reaching out to all four organizations to try to see if we can put together some kind of working group that can proceed and actually bring this to a reality.​” – by Chelsea Frajerman