ARTICLE
Miraflow, Soft Contact Lens Cleaner: Activity Against Acanthamoeba Spp Takeshi Kobayashi, Ph.D., Narumi Higuchi-Watanabe, M.S., Atsushi Shiraishi, M.D., Ph.D., Toshihiko Uno, M.D., Ph.D., and Yuichi Ohashi, M.D., Ph.D.
Objective: Miraflow is a cleaner for soft contact lens which contains 20% isopropyl alcohol. The purpose of this study was to determine the activity against Acanthamoeba trophozoites and cysts for Miraflow. In addition, to determine the activity of combined Miraflow and multipurpose solutions (MPSs) against Acanthamoeba cysts. Methods: Two simulated-use studies were conducted. The significance in the log reduction in the number of trophozoites and cysts of A. castellanii strains ATCC 50514 and ATCC 50370 or A. polyphaga ATCC 30461 after exposure to Miraflow alone was determined by the Spearman–Karber method. To examine the activity against Acanthamoeba of combined Miraflow and an MPS, the log reduction in the number of cysts after a 1-min exposure to Miraflow followed by a 4-hr exposure to MPS (ReNu fresh) was also determined. Results: Short-time exposure of 30 sec to Miraflow demonstrated activity against the Acanthamoeba trophozoites. However, a 1-min treatment was only relatively effective (1.1 log reduction) against the cysts of A. castellanii ATCC 50514, but no statistically significant reduction was observed for the cysts of the other 2 strains. The combined use with Miraflow and MPS demonstrated activity against the cysts, and a 3.0, 1.0, or 1.5 log reduction in the numbers was obtained for A. castellanii ATCC 50514, A. castellanii ATCC 50370, and A. polyphaga ATCC 30461, respectively. Conclusions: Exposure to combined Miraflow and MPS resulted in reductions in the number of Acanthamoeba cysts. Key Words: Acanthamoeba—Cysts—Miraflow—Multipurpose solutions— Combined activity. (Eye & Contact Lens 2015;41: 240–244)
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canthamoeba, a ubiquitous free-living amoeba, is known to cause painful and potentially blinding forms of keratitis. Acanthamoeba keratitis (AK) occurs most commonly in soft contact lens (SCL) wearers, and the estimated incidence of 90% of the reported cases may be attributed to SCL use.1 Approximately 1 in 30,000 SCL wearers develops AK,2 and the incidence of this From the Departments of Ophthalmology and Regenerative Medicine (T.K.), and Ophthalmology (N.H.-W., A.S., T.U., Y.O.), Ehime University School of Medicine, Toon, Japan. The Department of Ophthalmology, Ehime University School of Medicine, has received research funding from CIBA Vision (Tokyo, Japan). T. Kobayashi has received an honorarium from CIBA Vision for writing this article. The other authors have no conflicts of interest to disclose. Address correspondence to Takeshi Kobayashi, Ph.D., Department of Ophthalmology and Regenerative Medicine, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan; e-mail: [email protected] Accepted September 30, 2014. DOI: 10.1097/ICL.0000000000000112
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condition has increased in recent years.3 A national survey performed jointly by the Japanese Contact Lens Society and Ocular Infection Society showed that Pseudomonas aeruginosa and Acanthamoeba were the leading causes of serious cases of contact lens (CL)-related keratitis in Japan.4 Acanthamoeba can exist in two forms during its life cycle; the motile and metabolically active trophozoite, and the dormant cysts that form as a protective response to environmental stress such as starvation and rapid changes in pH, temperature, and osmolality. The cysts have a double wall containing acid-insoluble proteins and cellulose,5 and they are resistant to desiccation, ultraviolet radiation, and chemical agents. Careful cleaning and disinfection are needed for reusable SCLs with frequent planned-replacements. Although various cleaning and/or disinfecting solutions are commercially available, multipurpose solutions (MPSs) that can be used for cleaning, rinsing, and disinfecting are most frequently used in routine care of SCLs. Numerous studies have been performed evaluating the effects of commercial SCL disinfectant solutions on Acanthamoeba, and it was found that Acanthamoeba have high resistance to some of MPSs and can survive in some of SCL disinfectants and cleaners for long periods.6–14 However, several MPSs have possible disinfection efficacy against Acanthamoeba.15 In support of this resistance, Larkin et al.16 reported that Acanthamoeba was detected in 7% of the CL cases of 102 asymptomatic lens wearers who used MPS, and our recent study showed that Acanthamoeba contamination was found in 3 of the 33 lens cases of healthy adult SCL wearers who had used MPSs for daily lens care.17 It may be possible to gain additional efficacy against Acanthamoeba through the use of an adjunct product such as Miraflow (CIBA Vision, Tokyo, Japan). Miraflow is a cleaner for SCLs that contains 20% isopropyl alcohol, 15% Pluronic F-127, and 10% Miranol H2M.18 In the standard protocol, the SCL is rubbed with two or three drops of Miraflow, and the lens is rinsed and stored in a MPS. Miraflow would be expected to have some Acanthamoeba activity, because 20% isopropyl alcohol has been reported to be effective against Acanthamoeba trophozoites and cysts.19 Ghajar et al.20 showed that Miraflow showed some activity against trophozoites and cysts of A. castellanii strain ATCC 30234. Connor et al.21 also showed in a laboratory study that the number of A. culbertsoni cysts on SCLs was reduced when they were soaked in Miraflow. These studies suggested that Miraflow showed antiAcanthamoeba activity; however, both investigations used only a single strain of Acanthamoeba, namely, A. castellanii or A. culbertsoni. Thus, these results may not apply to other strains because it has been shown that the sensitivities to disinfectants Eye & Contact Lens Volume 41, Number 4, July 2015
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Eye & Contact Lens Volume 41, Number 4, July 2015 were different among the different Acanthamoeba species and strains.6,9,22 Therefore, it is helpful to examine the disinfectant activity against multiple strains of Acanthamoeba spp isolated from patients with keratitis. Thus, the purpose of this study was determine the activity of Miraflow against 2 strains of A. castellanii, ATCC 50514 and 50370, and 1 strain of A. polyphaga, ATCC 30461, all of which were isolated from AK patients. In addition, the activity of Miraflow combined with a MPS was investigated for activity against cysts of the three Acanthamoeba strains.
MATERIALS AND METHODS Acanthamoeba Trophozoites and Cysts We used A. castellanii, strains ATCC 50514 and ATCC 50370, and A. polyphaga, strain ATCC 30461, isolated from patients with AK for this study. Trophozoites were cultured in a peptone-yeast extract-glucose (PYG) medium (ATCC medium 712) in tissue culture flasks (Becton Dickinson, Tokyo, Japan) at 25°C. Encystment was induced by transferring the trophozoites from PYG medium to Neff’s constant-pH encystment medium and incubating the trophozoites for at least 2 weeks at 25°C.6,23 All procedures involving the organisms were carried out in biosafety level 2 laboratories.
Miraflow Activity Against Acanthamoeba Trophozoites and Cysts Precultured trophozoites or cysts were collected by scraping the bottom of culture flasks. After centrifugation (150g for 10 min), the organisms were suspended in 1/4 Ringer’s solution (Nihon Pharmaceutical, Tokyo, Japan) at a concentration of 5·106 organisms per millimeter. One milliliter of Miraflow was added to 10 mL of amoeba suspension, therefore the final concentration was 5·104 organisms per millimeter for the experimental solution, and 1 mL of 1/4 Ringer’s solution was used instead of Miraflow in the control solution. Subsequently, each sample was incubated at 25°C for 30 sec, 1 min, 5 min, 10 min, or 1 hr for trophozoites or 1 min, 5 min, 10 min, 15 min, 30 min, or 1 hr for cysts in 15-mL conical tubes (Sumitomo Bakelite, Tokyo, Japan). Immediately after the incubation period, the samples were diluted with 1/4 Ringer’s solution at a ratio of 9 parts 1/4 Ringer’s solution to 1 part test solution to stop the disinfecting reaction. In preliminary experiments, we determined the conditions required to block the antiAcanthamoeba activity of Miraflow. Ten milliliters of a 10-fold dilution of Miraflow was added to 10 mL of A. castellanii (ATCC 50370) trophozoite suspension (5·106/mL), and incubated at 25°C for 1 hr. Under these conditions, no reduction was observed. Thus, we confirmed that the viability of Acanthamoeba trophozoites was not affected by exposure to a 10-fold dilution of Miraflow (1 hr). After a dilution with 1/4 Ringer’s solution, the samples were washed by centrifugation and resuspended in 10 mL of 1/4 Ringer’s solution, and then centrifuged again and finally resuspended in 10 mL of PYG medium. Tenfold serial dilution of each amoebal suspension was made with PYG medium resulting in 4 dilutions with theoretical, maximum final concentrations of 5·103, 5·102, 5·101, and 5·100 amoeba per millimeter. Four 200-mL aliquots of each dilution were transferred to separate wells of a 96-well plate (Corning International, Tokyo, Japan), so that each dilution could be tested in quadruplicate. The 96-well plates were incubated at © 2015 Contact Lens Association of Ophthalmologists
Miraflow, SCL Cleaner 25°C. Samples containing trophozoites were incubated for 1 week, and those containing cysts were incubated for 3 weeks. At the end of the incubation period, amoebal growth in the wells was confirmed by phase contrast microscopy. The wells containing amoebal growth were counted, and the number of surviving organisms in each test solution was calculated using the Spearman–Karber equation as described in detail.6,24 The decrease in the number of organisms in each test solution was determined by subtracting the number in an experimental sample from the number of organisms in the control sample (1/4 Ringer’s solution). The number was expressed as a log reduction value.
Combined Miraflow and Multipurpose Solution Activity Against Acanthamoeba Cysts
One milliliter of Miraflow was added to 10 mL of Acanthamoeba cysts’ suspension to a final concentration of 5·104 organisms per millimeter. For the control solution, 1 mL of 1/4 Ringer’s solution was used instead of the Miraflow solution. The suspension was incubated at 25°C for 1 min, and then each sample was diluted and washed twice with 1/4 Ringer’s solution as described. The washed sample was resuspended in 10 mL of MPS (ReNu Fresh; Bausch and Lomb Japan, Tokyo, Japan), and for non-MPS treatment control, 1/4 Ringer’s solution was used instead of MPS. Subsequently, each sample was incubated at 25° C for 4 hr. After the incubation, the samples were centrifuged and resuspended in 1 mL of 1/4 Ringer’s solution. The residual active ingredients of MPSs, that is, polyhexamethylene biguanide (PHMB), in the samples were neutralized by adding an equal volume of Dey-Engley neutralizing broth (Sigma, St Louis, MO).6,15,25,26 The neutralized sample was serially diluted and cultured in PYG, and the number of surviving organisms was determined as described. The decrease in the number of organisms in each test solution was determined by subtraction from the number of cysts before inoculation.
Statistical Analyses One-way analysis of variance and Dunnett’s tests were used to determine whether the changes in the number of live organisms were significant in the different groups, and a P,0.05 was taken to be statistically significant.
RESULTS Miraflow Activity Against Acanthamoeba Trophozoites To examine the activity of Miraflow alone against Acanthamoeba, the log reduction in the number of surviving Acanthamoeba was determined after exposure of Acanthamoeba trophozoites to Miraflow. The trophozoites of 2 A. castellanii strains (ATCC 50514 and ATCC 50370) and 1 A. polyphaga strain (ATCC 30461) were exposed to Miraflow from 30 sec to 1 hr (Fig. 1A–C). After a 30-sec exposure to Miraflow, no growth of A. castellanii strains was observed (below detection limits, .3 log unit reduction; Fig. 1A,B). For the A. polyphaga strain, a 1.9 log unit reduction in the number of viable trophozoites was found after a 30-sec treatment. The number was reduced to below the detection limit by a 10-min exposure (Fig. 1C). 241
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T. Kobayashi et al.
FIG. 1. Miraflow activity against Acanthamoeba trophozoites. The log unit reduction in the number of surviving trophozoites of A. castellanii strains ATCC 50514 (A) and ATCC 50370 (B), and A. polyphaga strain ATCC 30461 (C) after 30-sec, 1-min, 5-min, 10min, or 1-hr exposure to Miraflow is shown. ND indicates that no growth was detected after the treatment (below the detection limit). The error bars indicate standard error of the mean. One-way analysis of variance and Dunnett’s tests were used to compare the difference between the number of survived organisms in test samples and those in control samples (***P,0.001; n=4).
Miraflow Activity Against Acanthamoeba Cysts Cysts of A. castellanii strains ATCC 50514 and ATCC 50370 and A. polyphaga strain ATCC 30461 were exposed to Miraflow for 1 min to 1 hr (Fig. 2A–C). A time-dependent reduction in the number of viable Acanthamoeba cysts was observed for all three tested strains. A 1-min exposure to Miraflow reduced the number of cysts of A. castellanii strain ATCC 50514 by 1.1 log units (P,0.01), which was statistically significantly. However, no statistically significant reduction was observed for the other 2 strains after a 1-min exposure (Fig. 2B,C). The number of cysts of A. castellanii strain ATCC 50514 was decreased to below the detection limit by a 30-min exposure to Miraflow (Fig. 2A). However, longer exposures (1 hr) were needed for the cysts of the other 2 strains to reduce the number to below the detection limits (Fig. 2B,C). 242
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FIG. 2. Miraflow activity against Acanthamoeba cysts. The log unit reduction in the number of surviving cysts of A. castellanii strains ATCC 50514 (A) and ATCC 50370 (B), and A. polyphaga strain ATCC 30461 (C) after 1-min, 5-min, 10-min, 15-min, 30-min, or 1-hr exposure to Miraflow are shown. ND indicates that no growth was detected after the treatment (below the detection limit). The error bars represents the standard error of the mean. One-way analysis of variance and Dunnett’s tests were used to compare the differences between the number of surviving organisms in test samples and those in control samples (*P=0.01–0.05; **P=0.001–0.01; ***P,0.001; ns, not significant; n=4).
Combined Miraflow and Multipurpose Solution Activity Against Acanthamoeba Cysts To examine the activity of combined Miraflow and MPS against Acanthamoeba cysts, the log reduction in the number of surviving cysts after 1-min exposure to Miraflow followed by a 4-hr exposure to an MPS (ReNu Fresh; Bausch and Lomb) was determined (Fig. 3A–C). After a 1-min and 4-hr exposure to combined Miraflow and MPS, there was a statistically significant reduction in the number of viable cysts of the A. castellanii strain ATCC 50514 (P,0.001) (Fig. 3A) to below the detection limit (.3 log reduction). With Miraflow alone, there was a 1.6 log unit reduction, and with MPS alone, there was a 1 log unit reduction in the number of cysts (Fig. 3A). The activity against Acanthamoeba for the 2 solutions was also effective against the other 2 tested strains (Fig. 3B,C), with Eye & Contact Lens Volume 41, Number 4, July 2015
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Eye & Contact Lens Volume 41, Number 4, July 2015
FIG. 3. Combined Miraflow and MPS activity against Acanthamoeba cysts. The log unit reductions in the number of surviving cysts of A. castellanii strains ATCC 50514 (A) and ATCC 50370 (B), and A. polyphaga strain ATCC 30461 (C) after a 4-hr exposure to 1/4 Ringer’s solution (control), 1-min exposure to Miraflow, 4-hr exposure to MPS (ReNu fresh, Bausch and Lomb), or after 1-min exposure to Miraflow and 4-hr exposure to MPS (ReNu fresh; Bausch and Lomb) are shown. ND indicates that no growth was detected after treatment (under the detection limit). The error bars represents the standard error of the mean. One-way analysis of variance and Dunnett’s tests were used to compare the difference among the treatments (**P=0.001–0.01; ***P,0.001; ns, not significant; n=5). MPS, multipurpose solution.
a 1 log unit reduction in the number of cysts of A. castellanii strain ATCC 50370 and a 1.5 log unit reduction for A. polyphaga strain ATCC 30461. However, no statistically significant reduction for these strains by a 4-hr exposure to MPS alone was observed.
DISCUSSION Miraflow is a cleaner for CLs, and it has been demonstrated to be effective in removing protein deposits and lipid contaminations from hydrogel lenses.27 In addition, Miraflow contains 20% isopropyl alcohol, which is highly effective against a broad spectrum of organisms including fungi and protozoan species.28 Ajello and © 2015 Contact Lens Association of Ophthalmologists
Miraflow, SCL Cleaner Ajello29 reported that a CL cleaner that contains 16% isopropyl alcohol was effective against different microorganisms including bacterial and fungal isolates from corneal ulcers and from the conjunctiva of patients with conjunctivitis. Aksozek et al.19 investigated the sensitivity of Acanthamoeba cysts against various chemicals and reported that they were sensitive to 20% isopropyl alcohol. In an anti-Acanthamoeba activity test, we first examined Miraflow alone against 3 Acanthamoeba strains isolated from patients with keratitis, and we found that a 30-sec exposure to Miraflow alone showed activity against Acanthamoeba trophozoites, although A. polyphaga (ATCC 30461) trophozoites were more resistant. In standard SCL care, the duration of the cleaning step with a lens cleaner was supposed to be less than a minute, but our findings suggest that such a short time exposure to Miraflow could potentially provide some activity against Acanthamoeba trophozoites. However, no statistically significant reduction was observed by a 1-min exposure for the cysts of 2 of the 3 strains tested. However, the cysts were time-dependent and reduced by exposure to Miraflow from 1 min to 1 hr. The results of several studies found that the susceptibility to disinfectants was dependent of the Acanthamoeba strain.6,9,22 Thus, Ghajar et al.20 and Connor et al.21 reported that Miraflow demonstrated some activity against Acanthamoeba trophozoites and cysts in a simulated-use laboratory study. However, they did not investigate the differences in activity for different strains of Acanthamoeba. Our results showed that the Miraflow activity against Acanthamoeba were different for the three Acanthamoeba strains studied. Miraflow is a daily cleaner used before disinfecting. For example, rubbing with Miraflow followed by rinsing and disinfection with MPS may be one of the standard protocols. However, there have been no studies that examined the combination of Miraflow and MPS. Our results showed a .1 log unit reduction in the number of cysts by all 3 tested strains when a combined 1-min exposure to Miraflow followed by a 4-hr exposure to MPS was used. It is generally believed that Acanthamoeba cysts have a high resistance to different disinfectants because cysts have double walls that are not permeable to various chemicals other than small molecules such as water, oxygen, and carbon dioxide.19,30–32 Thus, Acanthamoeba cysts could be resistant to PHMB contained in MPSs. Because isopropyl alcohol can denature proteins, isopropyl alcohol in Miraflow may denature the proteins of the walls of the cysts,5 and the cysts might become more permeable to the disinfectant ingredients such as PHMB. Although thermal disinfection is effective against Acanthamoeba, it is not known whether heat would shorten the life of SCLs, and heat is not recommended for high water content SCLs.20 Therefore, chemical disinfections such as MPSs have become the major disinfecting system for SCLs. It has been shown that a povidone–iodine solution was relatively effective against Acanthamoeba cysts compared with MPSs,6,33 and our previous study also showed the efficacy of povidone– iodine solution against cysts including strains tested in this study.6 We found a 2.6 log unit reduction in the number of cysts of A. castellanii strain ATCC 50514 and a 1.1 log unit reduction in the number of cysts for ATCC 50370 with povidone–iodine. Our findings indicated that the activity against Acanthamoeba when 243
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Eye & Contact Lens Volume 41, Number 4, July 2015
T. Kobayashi et al. Miraflow and MPS were combined was equal to that of povidone– iodine disinfection. It is generally believed that inadequate lens case hygiene and failure to wash the hands before handling the SCLs are risk factors for AK.34 Several studies have reported Acanthamoeba contaminations of the storage cases of asymptomatic CL wearers, and the authors suggested that the coexistence of bacteria might support Acanthamoeba growth in the storage cases.16,35–37 Therefore, a cleaning step by Miraflow before disinfection may mitigate the contamination of the lens case and reduce the exposure risk of Acanthamoeba to SCL users. In conclusion, our findings suggest that activity against Acanthamoeba may be increased by the combined use of Miraflow and MPS but further testing involving the regimen per each product’s indicated use is required. REFERENCES 1. Radford CF, Lehmann OJ, Dart JK. Acanthamoeba keratitis: Multicentre survey in England 1992–6. National Acanthamoeba Keratitis Study Group. Br J Ophthalmol 1998;82:1387–1392. 2. Seal DV. Acanthamoeba keratitis update-incidence, molecular epidemiology and new drugs for treatment. Eye (Lond) 2003;17:893–905. 3. Thebpatiphat N, Hammersmith KM, Rocha FN, et al. Acanthamoeba keratitis: A parasite on the rise. Cornea 2007;26:701–706. 4. Uno T, Fukuda M, Ohashi Y, et al. Survey of severe contact lens-associated microbial keratitis in Japan [in Japanese]. Nihon Ganka Gakkai Zasshi 2011;115:107–115. 5. Weisman RA. Differentiation in Acanthamoeba castellanii. Annu Rev Microbiol 1976;30:189–219. 6. Kobayashi T, Gibbon L, Mito T, et al. Efficacy of commercial soft contact lens disinfectant solutions against acanthamoeba. Jpn J Ophthalmol 2011; 55:547–557. 7. Brandt FH, Ware DA, Visvesvara GS. Viability of acanthamoeba cysts in ophthalmic solutions. Appl Environ Microbiol 1989;55:1144–1146. 8. Nauheim RC, Brockman RJ, Stopak SS, et al. Survival of acanthamoeba in contact lens rinse solutions. Cornea 1990;9:290–293. 9. Silvany RE, Dougherty JM, McCulley JP, et al. The effect of currently available contact lens disinfection systems on Acanthamoeba castellanii and Acanthamoeba polyphaga. Ophthalmology 1990;97:286–290. 10. Kilvington S, Anthony Y, Davies DJ, et al. Effect of contact lens disinfectants against acanthamoeba cysts. Rev Infect Dis 1991;13(Suppl 5):S414– S415. 11. Silvany RE, Dougherty JM, McCulley JP. Effect of contact lens preservatives on acanthamoeba. Ophthalmology 1991;98:854–857. 12. Beattie TK, Seal DV, Tomlinson A, et al. Determination of amoebicidal activities of multipurpose contact lens solutions by using a most probable number enumeration technique. J Clin Microbiol 2003;41:2992–3000. 13. Borazjani RN, Kilvington S. Efficacy of multipurpose solutions against Acanthamoeba species. Cont Lens Anterior Eye 2005;28:169–175. 14. Lonnen J, Heaselgrave W, Nomachi M, et al. Disinfection efficacy and encystment rate of soft contact lens multipurpose solutions against acanthamoeba. Eye Contact Lens 2010;36:26–32. 15. Mowrey-McKee M, George M. Contact lens solution efficacy against Acanthamoeba castellani. Eye Contact Lens 2007;33:211–215. 16. Larkin DF, Kilvington S, Easty DL. Contamination of contact lens storage cases by acanthamoeba and bacteria. Br J Ophthalmol 1990;74:133–135.
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17. Shiraishi A, Kono D, Sawaguchi S, et al. Microbial contamination of soft contact lens storage cases and contact lens care. J Jpn Cont Lens Soc 2011; 53:S34. 18. Weisbarth RE, Henderson B. Hydrogel lens care regimens and patient education. In: Bennett ES, Weissman BA, eds. Clinical Contact Lens Practice. 2nd ed. Philadelphia, PA, Lippincott Williams & Wilkins, 2005, pp 381–419. 19. Aksozek A, McClellan K, Howard K, et al. Resistance of Acanthamoeba castellanii cysts to physical, chemical, and radiological conditions. J Parasitol 2002;88:621–623. 20. Ghajar M, Houlsby RD, Chavez G. Microbiological evaluation of miraflow. J Am Optom Assoc 1989;60:592–595. 21. Connor CG, Hopkins SL, Salisbury RD. Effectivity of contact lens disinfection systems against Acanthamoeba culbertsoni. Optom Vis Sci 1991;68: 138–141. 22. Niszl IA, Markus MB. Anti-acanthamoeba activity of contact lens solutions. Br J Ophthalmol 1998;82:1033–1038. 23. Neff R, Ray S, Benton W, et al. Induction of synchronous encystment (differentiation) in Acanthamoeba sp. Methods Cell Physiol 1964;1:55–83. 24. Hamilton MA, Russo RC, Thurston RV. Trimmed spearman karber method for estimating median lethal concentrations in toxicity bioassays. Environ Sci Technol 1977;11:714–719. 25. Sutton SV, Wrzosek T, Proud DW. Neutralization efficacy of dey-engley medium in testing of contact lens disinfecting solutions. J Appl Bacteriol 1991;70:351–354. 26. Santos L, Oliveira R, Oliveira ME, et al. Lens material and formulation of multipurpose solutions affects contact lens disinfection. Cont Lens Anterior Eye 2011;34:179–182. 27. Simmons PA, Edrington TB, Lao KF, et al. The efficacy of disinfection systems for in-office storage of hydrogel contact lenses. Int Contact Lens Clin 1996;23:94–98. 28. Takahashi K. Alcohol and aldehide-disinfection efficacy, how to use and caution. Iyaku J 1986;22:2229–2232. 29. Ajello L, Ajello M. A comparison of the antimicrobial spectra and kill rates of three contact lens care solutions: QuickCARE starting solution, renu multi-purpose solution, and opti-free rinsing, disinfecting, and storage solution. Int Contact Lens Clin 1995;22:156–164. 30. Khunkitti W, Lloyd D, Furr JR, et al. Acanthamoeba castellanii: Growth, encystment, excystment and biocide susceptibility. J Infect 1998;36:43–48. 31. Turner NA, Russell AD, Furr JR, et al. Emergence of resistance to biocides during differentiation of Acanthamoeba castellanii. J Antimicrob Chemother 2000;46:27–34. 32. Chávez-Munguía B, Omaña-Molina M, González-Lázaro M, et al. Ultrastructural study of encystation and excystation in Acanthamoeba castellanii. J Eukaryot Microbiol 2005;52:153–158. 33. Kilvington S. Antimicrobial efficacy of a povidone iodine (pi) and a onestep hydrogen peroxide contact lens disinfection system. Cont Lens Anterior Eye 2004;27:209–212. 34. Butcko V, McMahon TT, Joslin CE, et al. Microbial keratitis and the role of rub and rinsing. Eye Contact Lens 2007;33:421–423; discussion 424–425. 35. Mayo MS, Schlitzer RL, Ward MA, et al. Association of pseudomonas and serratia corneal ulcers with use of contaminated solutions. J Clin Microbiol 1987;25:1398–1400. 36. Stehr-Green JK, Bailey TM, Brandt FH, et al. Acanthamoeba keratitis in soft contact lens wearers. A case-control study. J Am Med Assoc 1987;258: 57–60. 37. Gray TB, Cursons RTM, Sherwan JF, et al. Acanthamoeba, bacterial, and fungal contamination of contact lens storage cases. Br J Ophthalmol 1995; 79:601–605.
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Miraflow, Soft Contact Lens Cleaner: Activity Against Acanthamoeba Spp Takeshi Kobayashi, Ph.D., Narumi Higuchi-Watanabe, M.S., Atsushi Shiraishi, M.D., Ph.D., Toshihiko Uno, M.D., Ph.D., and Yuichi Ohashi, M.D., Ph.D.
Objective: Miraflow is a cleaner for soft contact lens which contains 20% isopropyl alcohol. The purpose of this study was to determine the activity against Acanthamoeba trophozoites and cysts for Miraflow. In addition, to determine the activity of combined Miraflow and multipurpose solutions (MPSs) against Acanthamoeba cysts. Methods: Two simulated-use studies were conducted. The significance in the log reduction in the number of trophozoites and cysts of A. castellanii strains ATCC 50514 and ATCC 50370 or A. polyphaga ATCC 30461 after exposure to Miraflow alone was determined by the Spearman–Karber method. To examine the activity against Acanthamoeba of combined Miraflow and an MPS, the log reduction in the number of cysts after a 1-min exposure to Miraflow followed by a 4-hr exposure to MPS (ReNu fresh) was also determined. Results: Short-time exposure of 30 sec to Miraflow demonstrated activity against the Acanthamoeba trophozoites. However, a 1-min treatment was only relatively effective (1.1 log reduction) against the cysts of A. castellanii ATCC 50514, but no statistically significant reduction was observed for the cysts of the other 2 strains. The combined use with Miraflow and MPS demonstrated activity against the cysts, and a 3.0, 1.0, or 1.5 log reduction in the numbers was obtained for A. castellanii ATCC 50514, A. castellanii ATCC 50370, and A. polyphaga ATCC 30461, respectively. Conclusions: Exposure to combined Miraflow and MPS resulted in reductions in the number of Acanthamoeba cysts. Key Words: Acanthamoeba—Cysts—Miraflow—Multipurpose solutions— Combined activity. (Eye & Contact Lens 2015;41: 240–244)
A
canthamoeba, a ubiquitous free-living amoeba, is known to cause painful and potentially blinding forms of keratitis. Acanthamoeba keratitis (AK) occurs most commonly in soft contact lens (SCL) wearers, and the estimated incidence of 90% of the reported cases may be attributed to SCL use.1 Approximately 1 in 30,000 SCL wearers develops AK,2 and the incidence of this From the Departments of Ophthalmology and Regenerative Medicine (T.K.), and Ophthalmology (N.H.-W., A.S., T.U., Y.O.), Ehime University School of Medicine, Toon, Japan. The Department of Ophthalmology, Ehime University School of Medicine, has received research funding from CIBA Vision (Tokyo, Japan). T. Kobayashi has received an honorarium from CIBA Vision for writing this article. The other authors have no conflicts of interest to disclose. Address correspondence to Takeshi Kobayashi, Ph.D., Department of Ophthalmology and Regenerative Medicine, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan; e-mail: [email protected] Accepted September 30, 2014. DOI: 10.1097/ICL.0000000000000112
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condition has increased in recent years.3 A national survey performed jointly by the Japanese Contact Lens Society and Ocular Infection Society showed that Pseudomonas aeruginosa and Acanthamoeba were the leading causes of serious cases of contact lens (CL)-related keratitis in Japan.4 Acanthamoeba can exist in two forms during its life cycle; the motile and metabolically active trophozoite, and the dormant cysts that form as a protective response to environmental stress such as starvation and rapid changes in pH, temperature, and osmolality. The cysts have a double wall containing acid-insoluble proteins and cellulose,5 and they are resistant to desiccation, ultraviolet radiation, and chemical agents. Careful cleaning and disinfection are needed for reusable SCLs with frequent planned-replacements. Although various cleaning and/or disinfecting solutions are commercially available, multipurpose solutions (MPSs) that can be used for cleaning, rinsing, and disinfecting are most frequently used in routine care of SCLs. Numerous studies have been performed evaluating the effects of commercial SCL disinfectant solutions on Acanthamoeba, and it was found that Acanthamoeba have high resistance to some of MPSs and can survive in some of SCL disinfectants and cleaners for long periods.6–14 However, several MPSs have possible disinfection efficacy against Acanthamoeba.15 In support of this resistance, Larkin et al.16 reported that Acanthamoeba was detected in 7% of the CL cases of 102 asymptomatic lens wearers who used MPS, and our recent study showed that Acanthamoeba contamination was found in 3 of the 33 lens cases of healthy adult SCL wearers who had used MPSs for daily lens care.17 It may be possible to gain additional efficacy against Acanthamoeba through the use of an adjunct product such as Miraflow (CIBA Vision, Tokyo, Japan). Miraflow is a cleaner for SCLs that contains 20% isopropyl alcohol, 15% Pluronic F-127, and 10% Miranol H2M.18 In the standard protocol, the SCL is rubbed with two or three drops of Miraflow, and the lens is rinsed and stored in a MPS. Miraflow would be expected to have some Acanthamoeba activity, because 20% isopropyl alcohol has been reported to be effective against Acanthamoeba trophozoites and cysts.19 Ghajar et al.20 showed that Miraflow showed some activity against trophozoites and cysts of A. castellanii strain ATCC 30234. Connor et al.21 also showed in a laboratory study that the number of A. culbertsoni cysts on SCLs was reduced when they were soaked in Miraflow. These studies suggested that Miraflow showed antiAcanthamoeba activity; however, both investigations used only a single strain of Acanthamoeba, namely, A. castellanii or A. culbertsoni. Thus, these results may not apply to other strains because it has been shown that the sensitivities to disinfectants Eye & Contact Lens Volume 41, Number 4, July 2015
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Eye & Contact Lens Volume 41, Number 4, July 2015 were different among the different Acanthamoeba species and strains.6,9,22 Therefore, it is helpful to examine the disinfectant activity against multiple strains of Acanthamoeba spp isolated from patients with keratitis. Thus, the purpose of this study was determine the activity of Miraflow against 2 strains of A. castellanii, ATCC 50514 and 50370, and 1 strain of A. polyphaga, ATCC 30461, all of which were isolated from AK patients. In addition, the activity of Miraflow combined with a MPS was investigated for activity against cysts of the three Acanthamoeba strains.
MATERIALS AND METHODS Acanthamoeba Trophozoites and Cysts We used A. castellanii, strains ATCC 50514 and ATCC 50370, and A. polyphaga, strain ATCC 30461, isolated from patients with AK for this study. Trophozoites were cultured in a peptone-yeast extract-glucose (PYG) medium (ATCC medium 712) in tissue culture flasks (Becton Dickinson, Tokyo, Japan) at 25°C. Encystment was induced by transferring the trophozoites from PYG medium to Neff’s constant-pH encystment medium and incubating the trophozoites for at least 2 weeks at 25°C.6,23 All procedures involving the organisms were carried out in biosafety level 2 laboratories.
Miraflow Activity Against Acanthamoeba Trophozoites and Cysts Precultured trophozoites or cysts were collected by scraping the bottom of culture flasks. After centrifugation (150g for 10 min), the organisms were suspended in 1/4 Ringer’s solution (Nihon Pharmaceutical, Tokyo, Japan) at a concentration of 5·106 organisms per millimeter. One milliliter of Miraflow was added to 10 mL of amoeba suspension, therefore the final concentration was 5·104 organisms per millimeter for the experimental solution, and 1 mL of 1/4 Ringer’s solution was used instead of Miraflow in the control solution. Subsequently, each sample was incubated at 25°C for 30 sec, 1 min, 5 min, 10 min, or 1 hr for trophozoites or 1 min, 5 min, 10 min, 15 min, 30 min, or 1 hr for cysts in 15-mL conical tubes (Sumitomo Bakelite, Tokyo, Japan). Immediately after the incubation period, the samples were diluted with 1/4 Ringer’s solution at a ratio of 9 parts 1/4 Ringer’s solution to 1 part test solution to stop the disinfecting reaction. In preliminary experiments, we determined the conditions required to block the antiAcanthamoeba activity of Miraflow. Ten milliliters of a 10-fold dilution of Miraflow was added to 10 mL of A. castellanii (ATCC 50370) trophozoite suspension (5·106/mL), and incubated at 25°C for 1 hr. Under these conditions, no reduction was observed. Thus, we confirmed that the viability of Acanthamoeba trophozoites was not affected by exposure to a 10-fold dilution of Miraflow (1 hr). After a dilution with 1/4 Ringer’s solution, the samples were washed by centrifugation and resuspended in 10 mL of 1/4 Ringer’s solution, and then centrifuged again and finally resuspended in 10 mL of PYG medium. Tenfold serial dilution of each amoebal suspension was made with PYG medium resulting in 4 dilutions with theoretical, maximum final concentrations of 5·103, 5·102, 5·101, and 5·100 amoeba per millimeter. Four 200-mL aliquots of each dilution were transferred to separate wells of a 96-well plate (Corning International, Tokyo, Japan), so that each dilution could be tested in quadruplicate. The 96-well plates were incubated at © 2015 Contact Lens Association of Ophthalmologists
Miraflow, SCL Cleaner 25°C. Samples containing trophozoites were incubated for 1 week, and those containing cysts were incubated for 3 weeks. At the end of the incubation period, amoebal growth in the wells was confirmed by phase contrast microscopy. The wells containing amoebal growth were counted, and the number of surviving organisms in each test solution was calculated using the Spearman–Karber equation as described in detail.6,24 The decrease in the number of organisms in each test solution was determined by subtracting the number in an experimental sample from the number of organisms in the control sample (1/4 Ringer’s solution). The number was expressed as a log reduction value.
Combined Miraflow and Multipurpose Solution Activity Against Acanthamoeba Cysts
One milliliter of Miraflow was added to 10 mL of Acanthamoeba cysts’ suspension to a final concentration of 5·104 organisms per millimeter. For the control solution, 1 mL of 1/4 Ringer’s solution was used instead of the Miraflow solution. The suspension was incubated at 25°C for 1 min, and then each sample was diluted and washed twice with 1/4 Ringer’s solution as described. The washed sample was resuspended in 10 mL of MPS (ReNu Fresh; Bausch and Lomb Japan, Tokyo, Japan), and for non-MPS treatment control, 1/4 Ringer’s solution was used instead of MPS. Subsequently, each sample was incubated at 25° C for 4 hr. After the incubation, the samples were centrifuged and resuspended in 1 mL of 1/4 Ringer’s solution. The residual active ingredients of MPSs, that is, polyhexamethylene biguanide (PHMB), in the samples were neutralized by adding an equal volume of Dey-Engley neutralizing broth (Sigma, St Louis, MO).6,15,25,26 The neutralized sample was serially diluted and cultured in PYG, and the number of surviving organisms was determined as described. The decrease in the number of organisms in each test solution was determined by subtraction from the number of cysts before inoculation.
Statistical Analyses One-way analysis of variance and Dunnett’s tests were used to determine whether the changes in the number of live organisms were significant in the different groups, and a P,0.05 was taken to be statistically significant.
RESULTS Miraflow Activity Against Acanthamoeba Trophozoites To examine the activity of Miraflow alone against Acanthamoeba, the log reduction in the number of surviving Acanthamoeba was determined after exposure of Acanthamoeba trophozoites to Miraflow. The trophozoites of 2 A. castellanii strains (ATCC 50514 and ATCC 50370) and 1 A. polyphaga strain (ATCC 30461) were exposed to Miraflow from 30 sec to 1 hr (Fig. 1A–C). After a 30-sec exposure to Miraflow, no growth of A. castellanii strains was observed (below detection limits, .3 log unit reduction; Fig. 1A,B). For the A. polyphaga strain, a 1.9 log unit reduction in the number of viable trophozoites was found after a 30-sec treatment. The number was reduced to below the detection limit by a 10-min exposure (Fig. 1C). 241
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T. Kobayashi et al.
FIG. 1. Miraflow activity against Acanthamoeba trophozoites. The log unit reduction in the number of surviving trophozoites of A. castellanii strains ATCC 50514 (A) and ATCC 50370 (B), and A. polyphaga strain ATCC 30461 (C) after 30-sec, 1-min, 5-min, 10min, or 1-hr exposure to Miraflow is shown. ND indicates that no growth was detected after the treatment (below the detection limit). The error bars indicate standard error of the mean. One-way analysis of variance and Dunnett’s tests were used to compare the difference between the number of survived organisms in test samples and those in control samples (***P,0.001; n=4).
Miraflow Activity Against Acanthamoeba Cysts Cysts of A. castellanii strains ATCC 50514 and ATCC 50370 and A. polyphaga strain ATCC 30461 were exposed to Miraflow for 1 min to 1 hr (Fig. 2A–C). A time-dependent reduction in the number of viable Acanthamoeba cysts was observed for all three tested strains. A 1-min exposure to Miraflow reduced the number of cysts of A. castellanii strain ATCC 50514 by 1.1 log units (P,0.01), which was statistically significantly. However, no statistically significant reduction was observed for the other 2 strains after a 1-min exposure (Fig. 2B,C). The number of cysts of A. castellanii strain ATCC 50514 was decreased to below the detection limit by a 30-min exposure to Miraflow (Fig. 2A). However, longer exposures (1 hr) were needed for the cysts of the other 2 strains to reduce the number to below the detection limits (Fig. 2B,C). 242
Eye & Contact Lens Volume 41, Number 4, July 2015
FIG. 2. Miraflow activity against Acanthamoeba cysts. The log unit reduction in the number of surviving cysts of A. castellanii strains ATCC 50514 (A) and ATCC 50370 (B), and A. polyphaga strain ATCC 30461 (C) after 1-min, 5-min, 10-min, 15-min, 30-min, or 1-hr exposure to Miraflow are shown. ND indicates that no growth was detected after the treatment (below the detection limit). The error bars represents the standard error of the mean. One-way analysis of variance and Dunnett’s tests were used to compare the differences between the number of surviving organisms in test samples and those in control samples (*P=0.01–0.05; **P=0.001–0.01; ***P,0.001; ns, not significant; n=4).
Combined Miraflow and Multipurpose Solution Activity Against Acanthamoeba Cysts To examine the activity of combined Miraflow and MPS against Acanthamoeba cysts, the log reduction in the number of surviving cysts after 1-min exposure to Miraflow followed by a 4-hr exposure to an MPS (ReNu Fresh; Bausch and Lomb) was determined (Fig. 3A–C). After a 1-min and 4-hr exposure to combined Miraflow and MPS, there was a statistically significant reduction in the number of viable cysts of the A. castellanii strain ATCC 50514 (P,0.001) (Fig. 3A) to below the detection limit (.3 log reduction). With Miraflow alone, there was a 1.6 log unit reduction, and with MPS alone, there was a 1 log unit reduction in the number of cysts (Fig. 3A). The activity against Acanthamoeba for the 2 solutions was also effective against the other 2 tested strains (Fig. 3B,C), with Eye & Contact Lens Volume 41, Number 4, July 2015
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Eye & Contact Lens Volume 41, Number 4, July 2015
FIG. 3. Combined Miraflow and MPS activity against Acanthamoeba cysts. The log unit reductions in the number of surviving cysts of A. castellanii strains ATCC 50514 (A) and ATCC 50370 (B), and A. polyphaga strain ATCC 30461 (C) after a 4-hr exposure to 1/4 Ringer’s solution (control), 1-min exposure to Miraflow, 4-hr exposure to MPS (ReNu fresh, Bausch and Lomb), or after 1-min exposure to Miraflow and 4-hr exposure to MPS (ReNu fresh; Bausch and Lomb) are shown. ND indicates that no growth was detected after treatment (under the detection limit). The error bars represents the standard error of the mean. One-way analysis of variance and Dunnett’s tests were used to compare the difference among the treatments (**P=0.001–0.01; ***P,0.001; ns, not significant; n=5). MPS, multipurpose solution.
a 1 log unit reduction in the number of cysts of A. castellanii strain ATCC 50370 and a 1.5 log unit reduction for A. polyphaga strain ATCC 30461. However, no statistically significant reduction for these strains by a 4-hr exposure to MPS alone was observed.
DISCUSSION Miraflow is a cleaner for CLs, and it has been demonstrated to be effective in removing protein deposits and lipid contaminations from hydrogel lenses.27 In addition, Miraflow contains 20% isopropyl alcohol, which is highly effective against a broad spectrum of organisms including fungi and protozoan species.28 Ajello and © 2015 Contact Lens Association of Ophthalmologists
Miraflow, SCL Cleaner Ajello29 reported that a CL cleaner that contains 16% isopropyl alcohol was effective against different microorganisms including bacterial and fungal isolates from corneal ulcers and from the conjunctiva of patients with conjunctivitis. Aksozek et al.19 investigated the sensitivity of Acanthamoeba cysts against various chemicals and reported that they were sensitive to 20% isopropyl alcohol. In an anti-Acanthamoeba activity test, we first examined Miraflow alone against 3 Acanthamoeba strains isolated from patients with keratitis, and we found that a 30-sec exposure to Miraflow alone showed activity against Acanthamoeba trophozoites, although A. polyphaga (ATCC 30461) trophozoites were more resistant. In standard SCL care, the duration of the cleaning step with a lens cleaner was supposed to be less than a minute, but our findings suggest that such a short time exposure to Miraflow could potentially provide some activity against Acanthamoeba trophozoites. However, no statistically significant reduction was observed by a 1-min exposure for the cysts of 2 of the 3 strains tested. However, the cysts were time-dependent and reduced by exposure to Miraflow from 1 min to 1 hr. The results of several studies found that the susceptibility to disinfectants was dependent of the Acanthamoeba strain.6,9,22 Thus, Ghajar et al.20 and Connor et al.21 reported that Miraflow demonstrated some activity against Acanthamoeba trophozoites and cysts in a simulated-use laboratory study. However, they did not investigate the differences in activity for different strains of Acanthamoeba. Our results showed that the Miraflow activity against Acanthamoeba were different for the three Acanthamoeba strains studied. Miraflow is a daily cleaner used before disinfecting. For example, rubbing with Miraflow followed by rinsing and disinfection with MPS may be one of the standard protocols. However, there have been no studies that examined the combination of Miraflow and MPS. Our results showed a .1 log unit reduction in the number of cysts by all 3 tested strains when a combined 1-min exposure to Miraflow followed by a 4-hr exposure to MPS was used. It is generally believed that Acanthamoeba cysts have a high resistance to different disinfectants because cysts have double walls that are not permeable to various chemicals other than small molecules such as water, oxygen, and carbon dioxide.19,30–32 Thus, Acanthamoeba cysts could be resistant to PHMB contained in MPSs. Because isopropyl alcohol can denature proteins, isopropyl alcohol in Miraflow may denature the proteins of the walls of the cysts,5 and the cysts might become more permeable to the disinfectant ingredients such as PHMB. Although thermal disinfection is effective against Acanthamoeba, it is not known whether heat would shorten the life of SCLs, and heat is not recommended for high water content SCLs.20 Therefore, chemical disinfections such as MPSs have become the major disinfecting system for SCLs. It has been shown that a povidone–iodine solution was relatively effective against Acanthamoeba cysts compared with MPSs,6,33 and our previous study also showed the efficacy of povidone– iodine solution against cysts including strains tested in this study.6 We found a 2.6 log unit reduction in the number of cysts of A. castellanii strain ATCC 50514 and a 1.1 log unit reduction in the number of cysts for ATCC 50370 with povidone–iodine. Our findings indicated that the activity against Acanthamoeba when 243
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Eye & Contact Lens Volume 41, Number 4, July 2015
T. Kobayashi et al. Miraflow and MPS were combined was equal to that of povidone– iodine disinfection. It is generally believed that inadequate lens case hygiene and failure to wash the hands before handling the SCLs are risk factors for AK.34 Several studies have reported Acanthamoeba contaminations of the storage cases of asymptomatic CL wearers, and the authors suggested that the coexistence of bacteria might support Acanthamoeba growth in the storage cases.16,35–37 Therefore, a cleaning step by Miraflow before disinfection may mitigate the contamination of the lens case and reduce the exposure risk of Acanthamoeba to SCL users. In conclusion, our findings suggest that activity against Acanthamoeba may be increased by the combined use of Miraflow and MPS but further testing involving the regimen per each product’s indicated use is required. REFERENCES 1. Radford CF, Lehmann OJ, Dart JK. Acanthamoeba keratitis: Multicentre survey in England 1992–6. National Acanthamoeba Keratitis Study Group. Br J Ophthalmol 1998;82:1387–1392. 2. Seal DV. Acanthamoeba keratitis update-incidence, molecular epidemiology and new drugs for treatment. Eye (Lond) 2003;17:893–905. 3. Thebpatiphat N, Hammersmith KM, Rocha FN, et al. Acanthamoeba keratitis: A parasite on the rise. Cornea 2007;26:701–706. 4. Uno T, Fukuda M, Ohashi Y, et al. Survey of severe contact lens-associated microbial keratitis in Japan [in Japanese]. Nihon Ganka Gakkai Zasshi 2011;115:107–115. 5. Weisman RA. Differentiation in Acanthamoeba castellanii. Annu Rev Microbiol 1976;30:189–219. 6. Kobayashi T, Gibbon L, Mito T, et al. Efficacy of commercial soft contact lens disinfectant solutions against acanthamoeba. Jpn J Ophthalmol 2011; 55:547–557. 7. Brandt FH, Ware DA, Visvesvara GS. Viability of acanthamoeba cysts in ophthalmic solutions. Appl Environ Microbiol 1989;55:1144–1146. 8. Nauheim RC, Brockman RJ, Stopak SS, et al. Survival of acanthamoeba in contact lens rinse solutions. Cornea 1990;9:290–293. 9. Silvany RE, Dougherty JM, McCulley JP, et al. The effect of currently available contact lens disinfection systems on Acanthamoeba castellanii and Acanthamoeba polyphaga. Ophthalmology 1990;97:286–290. 10. Kilvington S, Anthony Y, Davies DJ, et al. Effect of contact lens disinfectants against acanthamoeba cysts. Rev Infect Dis 1991;13(Suppl 5):S414– S415. 11. Silvany RE, Dougherty JM, McCulley JP. Effect of contact lens preservatives on acanthamoeba. Ophthalmology 1991;98:854–857. 12. Beattie TK, Seal DV, Tomlinson A, et al. Determination of amoebicidal activities of multipurpose contact lens solutions by using a most probable number enumeration technique. J Clin Microbiol 2003;41:2992–3000. 13. Borazjani RN, Kilvington S. Efficacy of multipurpose solutions against Acanthamoeba species. Cont Lens Anterior Eye 2005;28:169–175. 14. Lonnen J, Heaselgrave W, Nomachi M, et al. Disinfection efficacy and encystment rate of soft contact lens multipurpose solutions against acanthamoeba. Eye Contact Lens 2010;36:26–32. 15. Mowrey-McKee M, George M. Contact lens solution efficacy against Acanthamoeba castellani. Eye Contact Lens 2007;33:211–215. 16. Larkin DF, Kilvington S, Easty DL. Contamination of contact lens storage cases by acanthamoeba and bacteria. Br J Ophthalmol 1990;74:133–135.
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