1040-5488/14/9112-1440/0 VOL. 91, NO. 12, PP. 1440Y1445 OPTOMETRY AND VISION SCIENCE Copyright * 2014 American Academy of Optometry
ORIGINAL ARTICLE
Effects of Contact Lens Solution Disinfectants against Filamentous Fungi Yan Xu*, Yi He†, Lutan Zhou*, Chuanwen Gao*, Shengtao Sun*, Xiaochong Wang‡, and Guangren Pang ABSTRACT Purpose. To assess and compare the antifungal activity of polyhexamethylene biguanide (PHMB), thimerosal, cetylpyridinium chloride, and chlorhexidine, which are disinfectants used in multipurpose disinfectant solutions (MPDSs) against ocular pathogenic Fusarium solani and Aspergillus flavus isolates in vitro. Methods. The in vitro activity of PHMB, thimerosal, cetylpyridinium chloride, and chlorhexidine was assessed against 40 isolates of ocular pathogenic fungi that included 24 F. solani and 16 A. flavus isolates. The strains were tested by broth dilution antifungal susceptibility testing of filamentous fungi approved by the CLSI (Clinical and Laboratory Standards Institute) M38-A document. Results. MIC90 (minimum inhibitory concentration for 90% of the organisms) values of PHMB were 4 and 16 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of thimerosal were 0.0313 and 0.0625 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of cetylpyridinium chloride were 2 and 2 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of chlorhexidine were 32 and 32 Kg/mL for F. solani and A. flavus, respectively. Conclusions. As a disinfectant used in MPDSs, thimerosal showed the highest levels of antimicrobial activity against ocular pathogenic F. solani and A. flavus isolates. The concentrations of PHMB (0.0001%), cetylpyridinium chloride (0.00014%), and chlorhexidine (0.003%) in MPDSs are sublethal levels for ocular pathogenic F. solani and A. flavus isolates. Although multiple ingredients within MPDSs play a role in antimicrobial efficacy, antimicrobial activity may be significantly influenced by the disinfectants used in the solution formulations. (Optom Vis Sci 2014;91:1440Y1445) Key Words: polyhexamethylene biguanide, thimerosal, cetylpyridinium chloride, chlorhexidine, contact lensYrelated fungal keratitis, contact lens solution, Fusarium solani, Aspergillus flavus
C
ontact lensYrelated fungal keratitis occurs at a relatively low rate but stands out as a potentially sight-threatening event. The recent increase in the rate of contact lensYrelated Fusarium keratitis and multipurpose disinfectant solutions (MPDSs) becoming a key element in a worldwide outbreak of fungal keratitis have resulted in a renewed investigation into the possible contamination or inadequate disinfecting
*MD † BD ‡ PhD Laboratory of Ocular Pharmacology (YX, GP), Cornea Service (YH), Laboratory of Ocular Microbiology (SS), Henan Eye Institute and Henan Eye Hospital, Department of Ophthalmology, Henan Provincial People’s Hospital, Zhengzhou, Henan, China; Department of Optometry, Zhengzhou Railway Vocational and Technical College, Zhengzhou, Henan, China (LZ); Department of Ophthalmology, Zhengzhou Second Hospital, Zhengzhou, Henan, China (CG); and National University Hospital, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore (XW).
efficacy of MPDSs.1Y5 The most commonly described species related to contact lens fungal keratitis are Fusarium and Aspergillus species.3Y5 A noteworthy finding from some studies is a difference in the behavior of a standard strain (i.e., American Type Culture Collection [ATCC]) of Fusarium species compared with actual clinical isolates obtained from infected corneas. Under specific conditions, the clinical isolates are found to have a higher potential to survive and, in fact, thrive compared with the ATCC counterpart.1,2,6Y9 Although disinfectants are commonly added to MPDSs, scant research has been conducted on their effects on ocular pathogenic Fusarium and Aspergillus species. Similarly, the antifungal activities of polyhexamethylene biguanide (PHMB), thimerosal, cetylpyridinium chloride, and chlorhexidine, which are used as contact lens disinfectants, have not been adequately assessed against ocular pathogenic Fusarium solani and Aspergillus flavus in vitro. In this study, we determine whether the four disinfectants have equivalent abilities to inhibit ocular pathogenic F. solani and A. flavus in vitro. The main potential clinical
Optometry and Vision Science, Vol. 91, No. 12, December 2014
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Contact Lens Solution Disinfectants and Filamentous FungiVXu et al.
implication of the work is providing better prophylaxis options for contact lensYrelated fungal keratitis.
METHODS Test Isolates Twenty-four F. solani and 16 A. flavus strains isolated from patients with fungal keratitis from the Henan Eye Institute and the Henan Eye Hospital in Zhengzhou, China, were investigated. These isolates were identified to the species level based on morphology by standard methods.10 Candida parapsilosis ATCC 22019 was used as a quality control for each test.
Inoculum Preparation Inocula were prepared in accordance with Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) document M38-A211and were as described previously.12 Simply, the isolates were passaged twice at an interval of 7 days on potato dextrose agar slants at 35-C for 7 days (A. flavus) or at 35-C for 72 hours and then at 26-C until day 7 (F. solani). Seven-day-old colony was covered with 1 mL of sterile 0.85% saline, and then suspension was made. The resulting mixture of conidia and hyphal fragments was withdrawn. After heavy particles were allowed to settle for 3 to 5 minutes, the upper homogeneous suspension was collected and mixed with a vortex mixer for 15 seconds. The turbidity of the supernatants was measured spectrophotometrically at a wavelength of 530 nm, and transmission was adjusted to 68 to 70% (F. solani) or 80 to 82% (A. flavus). These suspensions were diluted 1:50 in RPMI 1640 medium (with L-glutamine, without sodium bicarbonate, Gibco BRL-Life Technologies, Grand Island, NY). The 1:50 inoculum dilutions corresponded to twice the density needed, about 0.4 104 to 5 104 CFU (colony-forming units)/mL.
Antifungal Agents Polyhexamethylene biguanide (Dr. Ehrenstorfer, Germany), thimerosal (Yili Pharmaceutical Co Ltd, Beijing, China), cetylpyridinium chloride (Fluka), and chlorhexidine (Jiutai Pharmaceutical Co Ltd, Jinzhou, China) were studied. They were dissolved in 100% dimethyl sulfoxide, respectively. The stock solutions were prepared at concentrations of 400 Kg/mL for thimerosal, 1600 Kg/mL for
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PHMB and cetylpyridinium chloride, and 3200 Kg/mL for chlorhexidine. Drug dilutions were made in RPMI 1640 medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (Serva, Feinbochemica GmbH & Co, Germany). Final concentrations ranged from 0.0078 to 4 Kg/mL for thimerosal, from 0.0313 to 16 Kg/mL for PHMB and cetylpyridinium chloride, and from 0.5 to 256 Kg/mL for chlorhexidine.
Antifungal Susceptibility In vitro susceptibility testing was performed by broth microdilution assay, in accordance with the CLSI M38-A2 guidelines for filamentous fungi.11 The tests were performed in duplicate in 96-well flat-bottom microtitration plates. Each well was inoculated with 0.1 mL of the 2 conidial inoculum suspension. The growth control wells contained 0.1 mL of the corresponding diluted inoculum suspension and 0.1 mL of RPMI-1640 broth without antifungal agents (with dimethyl sulfoxide 2%). Quality control was tested in the same manner and was included in each test. After incubation at 35-C for 48 hours, the minimum inhibitory concentration (MIC) was determined as the lowest concentration of the four disinfectants that prevented any discernible growth. The MIC range and mode, the MIC for 50% of the strains tested (MIC50 value), and the MIC90 value were provided for the isolates with the SPSS statistical package. For calculation, any high off-scale MIC was converted to the next higher concentration.
RESULTS The in vitro activities of PHMB, thimerosal, cetylpyridinium chloride, and chlorhexidine against the isolates are summarized in Table 1. MIC90 values of PHMB were 4 and 16 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of thimerosal were 0.0313 and 0.0625 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of cetylpyridinium chloride were 2 and 2 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of chlorhexidine were 32 and 32 Kg/mL for F. solani and A. flavus, respectively.
DISCUSSION Contact lens wear has been implicated as a predisposing factor in fungal keratitis.1Y5 Because fungal keratitis is difficult to treat,
TABLE 1.
In vitro susceptibilities of ocular pathogenic F. solani and A. flavus isolates to four disinfectants in MPDSs MICs, Kg/mL Organism (no. isolates) and disinfectants F. solani (24) PHMB TMR CPC CHD A. flavus (16) PHMB TMR CPC CHD
Range
Mode
MIC50
MIC90
2Y8 0.0078Y0.0313 2Y4 8Y32
2 0.0156 2 16
2 0.0156 2 16
4 0.0313 2 32
8Y16 0.0313Y0.0625 1Y2 2Y32
16 0.0625 2 32
16 0.0625 2 32
16 0.0625 2 32
TMR, thimerosal; CPC, cetylpyridinium chloride; CHD, chlorhexidine. Optometry and Vision Science, Vol. 91, No. 12, December 2014
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1442 Contact Lens Solution Disinfectants and Filamentous FungiVXu et al.
prevention is the main point in it. Prevention of infection in contact lens wearers depends on selection of the appropriate MPDSs and patient compliance.1Y5 Multipurpose disinfectant solutions should have a broad spectrum of antimicrobial activity and minimal toxicity. These properties are significantly influenced by the disinfectants used in the solution formulations. The MPDSs on the market are composed of different disinfectants, and the antimicrobial efficacy of an MPDS is a critical factor to consider when prescribing a lens care system.1Y5 This study was undertaken to evaluate the in vitro activities of disinfectants PHMB, thimerosal, cetylpyridinium chloride, and chlorhexidine used in MPDSs against ocular pathogenic F. solani and A. flavus with the ultimate goal of providing better prophylaxis options for contact lensYrelated fungal keratitis. Polyhexamethylene biguanide is a main antiseptic component in the widely used MPDSs to date, and the PHMB-based MPDSs reach 60% of the US market and most of the Chinese market.2 In MPDSs, PHMB is most usually present at 1 Kg/mL (0.0001%), and rarely at 0.5 Kg/mL (0.00005%) or 5 Kg/mL (0.0005%).7,8,13Y16 Based on our data, when comparing the MIC90 values of PHMB with its concentrations in MPDSs, the concentration of PHMB (0.00005 to 0.0001%) is lower than its MIC90 value against ocular pathogenic F. solani and much lower than its MIC90 value against A. flavus, and the concentration of PHMB (0.0005%) is higher than its MIC90 value against F. solani and lower than its MIC90 value against A. flavus (Table 2). Our results were compared with those obtained by other in vitro studies. In the study by Rebong et al.,17 the MIC value of PHMB was 4 Kg/mL against a single isolate of Aspergillus fumigatus. In the study by Behrens-Baumann et al.,18 MIC values of PHMB ranged from 1.56 to 3.12 Kg/mL for the nine Fusarium isolates as well as for another three isolates of ocular infections including one Scedosporium apiospermum, one A. fumigatus, and one Rhizopus microsporus. In the study of Messick et al.,19 MIC values of PHMB for a single Aspergillus niger isolate recovered from a patient with fungal keratitis and a standard strain F. solani ATCC 44366 were 6.1 and 2.4 Kg/mL, respectively. Our present results indicate that PHMB’s concentration (0.0001%), the most commonly used in MPDSs, is a sublethal level for ocular pathogenic F. solani and A. flavus and may not protect the contact lens wearers from a possible F. solani
and A. flavus infection of the eye. It should be addressed that MPDSs are systems and contain excipients other than just the disinfectants in the formulation, and multiple ingredients within the systems play a role in antimicrobial efficacy. The activity of PHMB is dependent on solution formulation and MPDSs with identical concentrations of PHMB can behave differently.15 Most PHMB-based MPDSs contain EDTA (ethylenediaminetetraacetic acid) that can potentiate the action of disinfectants, surfactants that can interact with the membranes of bacteria, cellulose-based agents that can rapidly bind to PHMB reducing its chemical availability and bioactivity, and sodium and potassium chloride that can reduce the cationic active sites and thus cytotoxicity.7,8,13Y16,20 However, it must be noted that our results were accordant with some antifungal activity studies on PHMB-based MPDSs that formulated multiple ingredients. Hume et al.7 compared the efficacy of MPDSs against F. solani ATCC 36031 and recent clinical isolates. The investigation included Complete Moisture PLUS and ReNu Multiplus; both PHMB-based MPDSs contain EDTA and surfactants. They were unable to achieve a 1-log10 kill with the two PHMB-based MPDSs against 10 clinical isolates of F. solani (ISO 1472921 requires a 1.0-log reduction in colony-forming units for the fungal species). Ide et al.16 evaluated and compared the in vitro effectiveness of MPDS to inhibit fungal colonization of FDA (Food and Drug Administration) contact lens groups. They showed that fungi were more likely (43.7 vs. 18.6%, p = 0.062) to be recovered from the PHMB-based product (ReNu MultiPlus, 0.0001% PHMB) than from the alexidine-based product or POLYQUAD-based solution. They conclude that the antifungal activity of contaminated MPDSs may be insufficient to prevent fungal colonization of contact lens materials despite meeting or exceeding the FDA stand-alone test criteria. Colonized lens may increase the risk of fungal keratitis. Loss of disinfecting efficacy after lenses are soaked in PHMB products for various periods of time has been demonstrated in several studies. Loss of disinfection efficacy during the disinfection process appears the most likely initiator of sustained Fusarium contamination of the solution, lens, and case.22 Polyhexamethylene biguanide has been used for the treatment of Acanthamoeba keratitis at concentrations of 0.02 to 0.053% and for the treatment of soft contact lensYrelated Fusarium
TABLE 2.
Comparison of the concentrations of four disinfectants in MPDSs with their MIC90 values against ocular pathogenic F. solani and A. flavus Disinfectant concentrations in MPDSs, Kg/mL 0.00005% PHMB (0.5) 0.0001% PHMB (1) 0.0005% PHMB (5) 0.002% TMR (20) 0.005% TMR (50) 0.00014% CPC (1.4) 0.003% CHD (30) 0.006% CHD (60) 0.015% CHD (150)
MIC90 against F. solani, Kg/mL
Concentration/MIC90 value
MIC90 against A. flavus, Kg/mL
4 4 4 0.0313 0.0313 2 32 32 32
0.125 0.25 1.25 639 1597 0.7 0.9 1.9 4.7
16 16 16 0.0625 0.0625 2 32 32 32
Concentration/MIC90 value
TMR, thimerosal; CPC, cetylpyridinium chloride; CHD, chlorhexidine. Optometry and Vision Science, Vol. 91, No. 12, December 2014
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0.0313 0.0625 0.3125 320 800 0.7 0.9 1.9 4.7
Contact Lens Solution Disinfectants and Filamentous FungiVXu et al.
keratitis at a concentration of 0.02% without causing adverse effects.18,23 When comparing the concentrations of PHMB in eye drops with its concentrations in MPDSs, the concentrations of PHMB in eye drops are 40 to 500 times as high as its concentrations in MPDSs. Based on our experimental results and evidence from the above studies, we propose that PHMB’s concentrations in MPDSs should be appropriately increased within tolerable limits to enhance antimicrobial effects. Thimerosal is a preservative commonly used in ophthalmic solutions, topical medicine, and vaccines. It is also used in MPDSs.24Y27 In this study, MIC90 values of thimerosal were 0.0313 and 0.0625 Kg/mL for ocular pathogenic F. solani and A. flavus, respectively. Thimerosal showed the highest levels of antimicrobial activity against fungal species and proved to be the most effective disinfectant. Our results were consistent with those obtained by us in a former investigation. That study showed that thimerosal’s antifungal activity was significantly superior to those of amphotericin B and natamycin against 244 ocular pathogenic fungi in vitro.28 Our results were also consistent with those from another study in which the MPDS Hydrocare with thimerosal was highly effective in eliminating A. fumigatus (ATCC 36607) contamination.24 In MPDSs, thimerosal is present at 20 or 50 Kg/mL (0.002 or 0.005%).18,20 Based on our data, when comparing the MIC90 values of thimerosal with its concentrations in MPDSs, the concentration of thimerosal is 639 times or 1597 times as high as its MIC90 value against ocular pathogenic F. solani and 320 times or 800 times as high as its MIC90 value against A. flavus (Table 2). Therefore, both ocular pathogenic F. solani and A. flavus were found very susceptible to thimerosal concentrations much lower than those present in MPDS formulations. So far, there is a clinical finding that an adverse effect induced by thimerosal as a preservative in MPDSs is ocular delayed hypersensitivity.29Y31 Patients with delayed hypersensitivity to thimerosal may develop conjunctival hyperemia, corneal infiltrates, and intolerance to lens wear with the use of MPDSs containing thimerosal. Because of allergic reactions, thimerosal is less used today in MPDSs. Based on our experimental results, we propose that thimerosal’s concentrations in MPDSs should be markedly decreased within antimicrobial effects to decrease side effects. Cetylpyridinium chloride is the antiseptic component in the widely used mouthwashes and is also the antiseptic component in some MPDSs.32,33 Although previous studies have documented cetylpyridinium chloride activity versus Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa,33 here our study first reported cetylpyridinium chloride activity versus ocular pathogenic F. solani and A. flavus isolates in vitro. In MPDSs, cetylpyridinium chloride is present at 1.4 Kg/mL (0.00014%).33 Based on our data, when comparing the MIC90 values of cetylpyridinium chloride with its concentration in MCLDSs, the concentration of cetylpyridinium chloride is lower than its MIC90 values against ocular pathogenic F. solani and A. flavus isolates (Table 2). In this study, cetylpyridinium chloride had a much lower MIC90 value than PHMB against A. flavus isolates; it appeared to be more effective than PHMB against ocular pathogenic A. flavus isolates. Chlorhexidine is a commonly used preservative in eye preparations and is also the antiseptic component in some MPDSs.25Y27
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Although previous studies have documented chlorhexidine activity versus S. aureus, E. coli, P. aeruginosa, Candida albicans, and Acanthamoeba,26,27,34 here our study first reported chlorhexidine activity versus ocular pathogenic F. solani and A. flavus isolates in vitro. In MPDSs, chlorhexidine is most commonly present at 30 or 60 Kg/mL (0.003 or 0.006%) and rarely present at 150 Kg/ mL (0.015%).13,25Y27 Based on our data, when comparing the MIC90 values of chlorhexidine with its concentrations in MPDSs, the concentration of chlorhexidine 0.003% is lower than its MIC90 values against F. solani and A. flavus isolates, and the concentration of chlorhexidine 0.006 or 0.015% is 1.9 times or 4.7 times as high as its MIC90 values against F. solani and A. flavus isolates, respectively (Table 2). Our present results indicate that in MPDSs, 0.003% chlorhexidine is a sublethal level, but 0.006 and 0.015% chlorhexidine are overlethal levels for ocular pathogenic F. solani and A. flavus isolates. The experimental protocol used in this study represents a different approach from those taken by ISO 14729 where several sequential steps and a standard strain F. solani ATCC 36031 are used to determine an MPDS’ efficacy. This study’s design focused on the efficacy of disinfectants PHMB, thimerosal, cetylpyridinium chloride, and chlorhexidine against ocular pathogenic F. solani and A. flavus isolates. F. solani is one of the most common causes of fungal keratitis and, therefore, is used as a challenge organism in microbiology disinfectant tests.21 We contributed additional data (MIC90 values) for the previously rare tested ocular pathogenic F. solani and A. flavus isolates. A notable result was that ocular pathogenic F. solani isolates tested in our investigation appeared to be less susceptible to PHMB than the ATCC strain tested in other investigations. Our result was in relative concordance with some studies.1,2,6Y9 In addition, A. flavus has emerged as a predominant ocular fungal pathogen12,35,36 but is not used as a challenge organism in microbiology disinfectant tests. Our studies provided additional evidence to determine the effectiveness of PHMB against A. flavus. In our study, the MIC90 value of PHMB was 16 Kg/mL for A. flavus isolates and A. flavus appeared to be less susceptible than F. solani to the antifungal action of PHMB. Our study, like those of others,6Y9 revealed inadequacies in the testing procedures recommended by ISO 14729, where only one Fusarium isolate is used to challenge the disinfectants.21 The use of a single laboratory strain and a single source of inoculum may provide a false sense of assurance that the disinfecting solution is effective.9 We think that the test could be improved by the use of both laboratory strains and multiple clinical strains to reduce the risk of fungal keratitis associated with contact lens wear. In conclusion, in this study, as a disinfectant used in MPDSs, thimerosal showed the highest levels of antimicrobial activity against ocular pathogenic F. solani and A. flavus isolates. The concentrations of PHMB (0.0001%), cetylpyridinium chloride (0.00014%), and chlorhexidine (0.003%) in MPDSs are sublethal levels for ocular pathogenic F. solani and A. flavus isolates. Although multiple ingredients within MPDSs play a role in antimicrobial efficacy, antimicrobial activity may be significantly influenced by the disinfectants used in the solution formulations.
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1444 Contact Lens Solution Disinfectants and Filamentous FungiVXu et al.
ACKNOWLEDGMENTS This work was supported by the National Natural Science Fund of China (81241033), Fund of Bureau of Health of Henan Province (201002012), and Fund of Bureau of Science and Technology of Henan Province (0641130305). Received March 9, 2014; accepted June 16, 2014.
REFERENCES 1. Levy B, Heiler D, Norton S. Report on testing from an investigation of Fusarium keratitis in contact lens wearers. Eye Contact Lens 2006;32:256Y61. 2. Levy B. Infectious keratitis: what have we learned? Eye Contact Lens 2007;33:418Y20. 3. Chang DC, Grant GB, O’Donnell K, Wannemuehler KA, NobleWang J, Rao CY, Jacobson LM, Crowell CS, Sneed RS, Lewis FM, Schaffzin JK, Kainer MA, Genese CA, Alfonso EC, Jones DB, Srinivasan A, Fridkin SK, Park BJ. Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution. JAMA 2006; 296:953Y63. 4. Donnio A, Van Nuoi DN, Catanese M, Desbois N, Ayeboua L, Merle H. Outbreak of keratomycosis attributable to Fusarium solani in the French West Indies. Am J Ophthalmol 2007;143:356Y8. 5. Khor WB, Aung T, Saw SM, Wong TY, Tambyah PA, Tan AL, Beuerman R, Lim L, Chan WK, Heng WJ, Lim J, Loh RS, Lee SB, Tan DT. An outbreak of Fusarium keratitis associated with contact lens wear in Singapore. JAMA 2006;295:2867Y73. 6. Zhang S, Ahearn DG, Stulting RD, Schwam BL, Simmons RB, Pierce GE, Crow SA, Jr. Differences among strains of the Fusarium oxysporum-F. solani complexes in their penetration of hydrogel contact lenses and subsequent susceptibility to multipurpose contact lens disinfection solutions. Cornea 2007;26:1249Y54. 7. Hume EB, Flanagan J, Masoudi S, Zhu H, Cole N, Willcox MD. Soft contact lens disinfection solution efficacy: clinical Fusarium isolates vs. ATCC 36031. Optom Vis Sci 2009;86:415Y9. 8. Kilvington S, Lam A, Nikolic M, Brady N. Resistance and growth of Fusarium species in contact lens disinfectant solutions. Optom Vis Sci 2013;90:430Y8. 9. Boost M, Lai S, Ma C, Cho P. Do multipurpose contact lens disinfecting solutions work effectively against non-FDA/ISO recommended strains of bacteria and fungi? Ophthalmic Physiol Opt 2010;30:12Y9. 10. Wang DL. Medical MycologyVGuide to Laboratory Examination. Beijing, China: People’s Medical Publishing House; 2005. 11. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. Document M38-A2. Wayne, PA: NCCLS; 2002. 12. Xu Y, Gao C, Li X, He Y, Zhou L, Pang G, Sun S. In vitro antifungal activity of silver nanoparticles against ocular pathogenic filamentous fungi. J Ocul Pharmacol Ther 2013;29:270Y4. 13. Hiti K, Walochnik J, Maria Haller-Schober E, Faschinger C, Aspock H. Efficacy of contact lens storage solutions against different Acanthamoeba strains. Cornea 2006;25:423Y7. 14. Kilvington S, Powell CH, Lam A, Lonnen J. Antimicrobial efficacy of multi-purpose contact lens disinfectant solutions following evaporation. Cont Lens Anterior Eye 2011;34:183Y7.
15. Santodomingo-Rubido J, Mori O, Kawaminami S. Cytotoxicity and antimicrobial activity of six multipurpose soft contact lens disinfecting solutions. Ophthalmic Physiol Opt 2006;26:476Y82. 16. Ide T, Miller D, Alfonso EC, O’Brien TP. Impact of contact lens group on antifungal efficacy of multipurpose disinfecting contact lens solutions. Eye Contact Lens 2008;34:151Y9. 17. Rebong RA, Santaella RM, Goldhagen BE, Majka CP, Perfect JR, Steinbach WJ, Afshari NA. Polyhexamethylene biguanide and calcineurin inhibitors as novel antifungal treatments for Aspergillus keratitis. Invest Ophthalmol Vis Sci 2011;52:7309Y15. 18. Behrens-Baumann W, Seibold M, Hofmuller W, Walter S, Haeberle H, Wecke T, Tammer I, Tintelnot K. Benefit of polyhexamethylene biguanide in Fusarium keratitis. Ophthalmic Res 2012;48:171Y6. 19. Messick CR, Pendland SL, Moshirfar M, Fiscella RG, Losnedahl KJ, Schriever CA, Schreckenberger PC. In-vitro activity of polyhexamethylene biguanide (PHMB) against fungal isolates associated with infective keratitis. J Antimicrob Chemother 1999; 44:297Y8. 20. Vehige JG, Simmons PA, Anger C, Graham R, Tran L, Brady N. Cytoprotective properties of carboxymethyl cellulose (CMC) when used prior to wearing contact lenses treated with cationic disinfecting agents. Eye Contact Lens 2003;29:177Y80. 21. International Organization for Standardization (ISO). Ophthalmic OpticsVContact Lens ProductsVMicrobial Requirements and Test Method for Products and Regimes for Hygienic Management of Contact Lenses: ISO 14729. Geneva, Switzerland: ISO; 2001. 22. Rosenthal RA, Dassanayake NL, Schlitzer RL, Schlech BA, Meadows DL, Stone RP. Biocide uptake in contact lenses and loss of fungicidal activity during storage of contact lenses. Eye Contact Lens 2006;32:262Y6. 23. Duguid IG, Dart JK, Morlet N, Allan BD, Matheson M, Ficker L, Tuft S. Outcome of Acanthamoeba keratitis treated with polyhexamethyl biguanide and propamidine. Ophthalmology 1997; 104:1587Y92. 24. Connor CG, Presley L, Finchum SM, Steel SA. The effectiveness of several current soft contact lens care systems against Aspergillus. CLAO J 1998;24:82Y4. 25. Imayasu M, Moriyama T, Ichijima H, Ohashi J, Petroll WM, Jester JV, Cavanagh HD. The effects of daily wear of rigid gas permeable contact lenses treated with contact lens care solutions containing preservatives on the rabbit cornea. CLAO J 1994; 20:183Y8. 26. Keeven J, Wrobel S, Portoles M, DeCicco BT. Evaluating the preservative effectiveness of RGP lens care solutions. CLAO J 1995; 21:238Y41. 27. Zanetti S, Fiori PL, Pinna A, Usai S, Carta F, Fadda G. Susceptibility of Acanthamoeba castellanii to contact lens disinfecting solutions. Antimicrob Agents Chemother 1995;39:1596Y8. 28. Xu Y, Pang G, Zhao D, Gao C, Zhou L, Sun S, Wang B. In vitro activity of thimerosal against ocular pathogenic fungi. Antimicrob Agents Chemother 2010;54:536Y9. 29. Wilson LA, McNatt J, Reitschel R. Delayed hypersensitivity to thimerosal in soft contact lens wearers. Ophthalmology 1981; 88:804Y9. 30. Mondino BJ, Salamon SM, Zaidman GW. Allergic and toxic reactions of soft contact lens wearers. Surv Ophthalmol 1982; 26:337Y44. 31. Wright P, Mackie I. Preservative-related problems in soft contact lens wearers. Trans Ophthalmol Soc U K 1982;102(Pt. 1):3Y6.
Optometry and Vision Science, Vol. 91, No. 12, December 2014
Copyright © American Academy of Optometry. Unauthorized reproduction of this article is prohibited.
Contact Lens Solution Disinfectants and Filamentous FungiVXu et al. 32. Yu ZJ, Huth SW. Cetylpyridinium chloride as an antimicrobial agent in ophthalmic compositions. US patent 7,578,996. August 25, 2009. 33. Chen JY, He T, Jia XH. [The sorption was observed on the active ingredient of contact lens care solution by germicidal test.]. Chin Med Equip News 2006;2:65Y7. 34. Zhu B, Liu N, Lin LY, Li PB, Yin T. [Study on germicidal efficacy and toxicity of multi-functional contact lens cleaning-disinfection solution.]. Int J Lab Med 2009;30:274Y6. 35. Xu Y, Zhao D, Gao C, Zhou L, Pang G, Sun S. In vitro activity of phenylmercuric acetate against ocular pathogenic fungi. J Antimicrob Chemother 2012;67:1941Y4.
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36. Xie L, Zhai H, Zhao J, Sun S, Shi W, Dong X. Antifungal susceptibility for common pathogens of fungal keratitis in Shandong Province, China. Am J Ophthalmol 2008;146:260Y5.
Yan Xu Laboratory of Ocular Pharmacology Henan Eye Institute and Henan Eye Hospital 7 Weiwu Rd Zhengzhou, 450003 China e-mail: [email protected]
Optometry and Vision Science, Vol. 91, No. 12, December 2014
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ORIGINAL ARTICLE
Effects of Contact Lens Solution Disinfectants against Filamentous Fungi Yan Xu*, Yi He†, Lutan Zhou*, Chuanwen Gao*, Shengtao Sun*, Xiaochong Wang‡, and Guangren Pang ABSTRACT Purpose. To assess and compare the antifungal activity of polyhexamethylene biguanide (PHMB), thimerosal, cetylpyridinium chloride, and chlorhexidine, which are disinfectants used in multipurpose disinfectant solutions (MPDSs) against ocular pathogenic Fusarium solani and Aspergillus flavus isolates in vitro. Methods. The in vitro activity of PHMB, thimerosal, cetylpyridinium chloride, and chlorhexidine was assessed against 40 isolates of ocular pathogenic fungi that included 24 F. solani and 16 A. flavus isolates. The strains were tested by broth dilution antifungal susceptibility testing of filamentous fungi approved by the CLSI (Clinical and Laboratory Standards Institute) M38-A document. Results. MIC90 (minimum inhibitory concentration for 90% of the organisms) values of PHMB were 4 and 16 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of thimerosal were 0.0313 and 0.0625 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of cetylpyridinium chloride were 2 and 2 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of chlorhexidine were 32 and 32 Kg/mL for F. solani and A. flavus, respectively. Conclusions. As a disinfectant used in MPDSs, thimerosal showed the highest levels of antimicrobial activity against ocular pathogenic F. solani and A. flavus isolates. The concentrations of PHMB (0.0001%), cetylpyridinium chloride (0.00014%), and chlorhexidine (0.003%) in MPDSs are sublethal levels for ocular pathogenic F. solani and A. flavus isolates. Although multiple ingredients within MPDSs play a role in antimicrobial efficacy, antimicrobial activity may be significantly influenced by the disinfectants used in the solution formulations. (Optom Vis Sci 2014;91:1440Y1445) Key Words: polyhexamethylene biguanide, thimerosal, cetylpyridinium chloride, chlorhexidine, contact lensYrelated fungal keratitis, contact lens solution, Fusarium solani, Aspergillus flavus
C
ontact lensYrelated fungal keratitis occurs at a relatively low rate but stands out as a potentially sight-threatening event. The recent increase in the rate of contact lensYrelated Fusarium keratitis and multipurpose disinfectant solutions (MPDSs) becoming a key element in a worldwide outbreak of fungal keratitis have resulted in a renewed investigation into the possible contamination or inadequate disinfecting
*MD † BD ‡ PhD Laboratory of Ocular Pharmacology (YX, GP), Cornea Service (YH), Laboratory of Ocular Microbiology (SS), Henan Eye Institute and Henan Eye Hospital, Department of Ophthalmology, Henan Provincial People’s Hospital, Zhengzhou, Henan, China; Department of Optometry, Zhengzhou Railway Vocational and Technical College, Zhengzhou, Henan, China (LZ); Department of Ophthalmology, Zhengzhou Second Hospital, Zhengzhou, Henan, China (CG); and National University Hospital, Yong Loo Lin School of Medicine, National University of Singapore, Republic of Singapore (XW).
efficacy of MPDSs.1Y5 The most commonly described species related to contact lens fungal keratitis are Fusarium and Aspergillus species.3Y5 A noteworthy finding from some studies is a difference in the behavior of a standard strain (i.e., American Type Culture Collection [ATCC]) of Fusarium species compared with actual clinical isolates obtained from infected corneas. Under specific conditions, the clinical isolates are found to have a higher potential to survive and, in fact, thrive compared with the ATCC counterpart.1,2,6Y9 Although disinfectants are commonly added to MPDSs, scant research has been conducted on their effects on ocular pathogenic Fusarium and Aspergillus species. Similarly, the antifungal activities of polyhexamethylene biguanide (PHMB), thimerosal, cetylpyridinium chloride, and chlorhexidine, which are used as contact lens disinfectants, have not been adequately assessed against ocular pathogenic Fusarium solani and Aspergillus flavus in vitro. In this study, we determine whether the four disinfectants have equivalent abilities to inhibit ocular pathogenic F. solani and A. flavus in vitro. The main potential clinical
Optometry and Vision Science, Vol. 91, No. 12, December 2014
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Contact Lens Solution Disinfectants and Filamentous FungiVXu et al.
implication of the work is providing better prophylaxis options for contact lensYrelated fungal keratitis.
METHODS Test Isolates Twenty-four F. solani and 16 A. flavus strains isolated from patients with fungal keratitis from the Henan Eye Institute and the Henan Eye Hospital in Zhengzhou, China, were investigated. These isolates were identified to the species level based on morphology by standard methods.10 Candida parapsilosis ATCC 22019 was used as a quality control for each test.
Inoculum Preparation Inocula were prepared in accordance with Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) document M38-A211and were as described previously.12 Simply, the isolates were passaged twice at an interval of 7 days on potato dextrose agar slants at 35-C for 7 days (A. flavus) or at 35-C for 72 hours and then at 26-C until day 7 (F. solani). Seven-day-old colony was covered with 1 mL of sterile 0.85% saline, and then suspension was made. The resulting mixture of conidia and hyphal fragments was withdrawn. After heavy particles were allowed to settle for 3 to 5 minutes, the upper homogeneous suspension was collected and mixed with a vortex mixer for 15 seconds. The turbidity of the supernatants was measured spectrophotometrically at a wavelength of 530 nm, and transmission was adjusted to 68 to 70% (F. solani) or 80 to 82% (A. flavus). These suspensions were diluted 1:50 in RPMI 1640 medium (with L-glutamine, without sodium bicarbonate, Gibco BRL-Life Technologies, Grand Island, NY). The 1:50 inoculum dilutions corresponded to twice the density needed, about 0.4 104 to 5 104 CFU (colony-forming units)/mL.
Antifungal Agents Polyhexamethylene biguanide (Dr. Ehrenstorfer, Germany), thimerosal (Yili Pharmaceutical Co Ltd, Beijing, China), cetylpyridinium chloride (Fluka), and chlorhexidine (Jiutai Pharmaceutical Co Ltd, Jinzhou, China) were studied. They were dissolved in 100% dimethyl sulfoxide, respectively. The stock solutions were prepared at concentrations of 400 Kg/mL for thimerosal, 1600 Kg/mL for
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PHMB and cetylpyridinium chloride, and 3200 Kg/mL for chlorhexidine. Drug dilutions were made in RPMI 1640 medium buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (Serva, Feinbochemica GmbH & Co, Germany). Final concentrations ranged from 0.0078 to 4 Kg/mL for thimerosal, from 0.0313 to 16 Kg/mL for PHMB and cetylpyridinium chloride, and from 0.5 to 256 Kg/mL for chlorhexidine.
Antifungal Susceptibility In vitro susceptibility testing was performed by broth microdilution assay, in accordance with the CLSI M38-A2 guidelines for filamentous fungi.11 The tests were performed in duplicate in 96-well flat-bottom microtitration plates. Each well was inoculated with 0.1 mL of the 2 conidial inoculum suspension. The growth control wells contained 0.1 mL of the corresponding diluted inoculum suspension and 0.1 mL of RPMI-1640 broth without antifungal agents (with dimethyl sulfoxide 2%). Quality control was tested in the same manner and was included in each test. After incubation at 35-C for 48 hours, the minimum inhibitory concentration (MIC) was determined as the lowest concentration of the four disinfectants that prevented any discernible growth. The MIC range and mode, the MIC for 50% of the strains tested (MIC50 value), and the MIC90 value were provided for the isolates with the SPSS statistical package. For calculation, any high off-scale MIC was converted to the next higher concentration.
RESULTS The in vitro activities of PHMB, thimerosal, cetylpyridinium chloride, and chlorhexidine against the isolates are summarized in Table 1. MIC90 values of PHMB were 4 and 16 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of thimerosal were 0.0313 and 0.0625 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of cetylpyridinium chloride were 2 and 2 Kg/mL for F. solani and A. flavus, respectively. MIC90 values of chlorhexidine were 32 and 32 Kg/mL for F. solani and A. flavus, respectively.
DISCUSSION Contact lens wear has been implicated as a predisposing factor in fungal keratitis.1Y5 Because fungal keratitis is difficult to treat,
TABLE 1.
In vitro susceptibilities of ocular pathogenic F. solani and A. flavus isolates to four disinfectants in MPDSs MICs, Kg/mL Organism (no. isolates) and disinfectants F. solani (24) PHMB TMR CPC CHD A. flavus (16) PHMB TMR CPC CHD
Range
Mode
MIC50
MIC90
2Y8 0.0078Y0.0313 2Y4 8Y32
2 0.0156 2 16
2 0.0156 2 16
4 0.0313 2 32
8Y16 0.0313Y0.0625 1Y2 2Y32
16 0.0625 2 32
16 0.0625 2 32
16 0.0625 2 32
TMR, thimerosal; CPC, cetylpyridinium chloride; CHD, chlorhexidine. Optometry and Vision Science, Vol. 91, No. 12, December 2014
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1442 Contact Lens Solution Disinfectants and Filamentous FungiVXu et al.
prevention is the main point in it. Prevention of infection in contact lens wearers depends on selection of the appropriate MPDSs and patient compliance.1Y5 Multipurpose disinfectant solutions should have a broad spectrum of antimicrobial activity and minimal toxicity. These properties are significantly influenced by the disinfectants used in the solution formulations. The MPDSs on the market are composed of different disinfectants, and the antimicrobial efficacy of an MPDS is a critical factor to consider when prescribing a lens care system.1Y5 This study was undertaken to evaluate the in vitro activities of disinfectants PHMB, thimerosal, cetylpyridinium chloride, and chlorhexidine used in MPDSs against ocular pathogenic F. solani and A. flavus with the ultimate goal of providing better prophylaxis options for contact lensYrelated fungal keratitis. Polyhexamethylene biguanide is a main antiseptic component in the widely used MPDSs to date, and the PHMB-based MPDSs reach 60% of the US market and most of the Chinese market.2 In MPDSs, PHMB is most usually present at 1 Kg/mL (0.0001%), and rarely at 0.5 Kg/mL (0.00005%) or 5 Kg/mL (0.0005%).7,8,13Y16 Based on our data, when comparing the MIC90 values of PHMB with its concentrations in MPDSs, the concentration of PHMB (0.00005 to 0.0001%) is lower than its MIC90 value against ocular pathogenic F. solani and much lower than its MIC90 value against A. flavus, and the concentration of PHMB (0.0005%) is higher than its MIC90 value against F. solani and lower than its MIC90 value against A. flavus (Table 2). Our results were compared with those obtained by other in vitro studies. In the study by Rebong et al.,17 the MIC value of PHMB was 4 Kg/mL against a single isolate of Aspergillus fumigatus. In the study by Behrens-Baumann et al.,18 MIC values of PHMB ranged from 1.56 to 3.12 Kg/mL for the nine Fusarium isolates as well as for another three isolates of ocular infections including one Scedosporium apiospermum, one A. fumigatus, and one Rhizopus microsporus. In the study of Messick et al.,19 MIC values of PHMB for a single Aspergillus niger isolate recovered from a patient with fungal keratitis and a standard strain F. solani ATCC 44366 were 6.1 and 2.4 Kg/mL, respectively. Our present results indicate that PHMB’s concentration (0.0001%), the most commonly used in MPDSs, is a sublethal level for ocular pathogenic F. solani and A. flavus and may not protect the contact lens wearers from a possible F. solani
and A. flavus infection of the eye. It should be addressed that MPDSs are systems and contain excipients other than just the disinfectants in the formulation, and multiple ingredients within the systems play a role in antimicrobial efficacy. The activity of PHMB is dependent on solution formulation and MPDSs with identical concentrations of PHMB can behave differently.15 Most PHMB-based MPDSs contain EDTA (ethylenediaminetetraacetic acid) that can potentiate the action of disinfectants, surfactants that can interact with the membranes of bacteria, cellulose-based agents that can rapidly bind to PHMB reducing its chemical availability and bioactivity, and sodium and potassium chloride that can reduce the cationic active sites and thus cytotoxicity.7,8,13Y16,20 However, it must be noted that our results were accordant with some antifungal activity studies on PHMB-based MPDSs that formulated multiple ingredients. Hume et al.7 compared the efficacy of MPDSs against F. solani ATCC 36031 and recent clinical isolates. The investigation included Complete Moisture PLUS and ReNu Multiplus; both PHMB-based MPDSs contain EDTA and surfactants. They were unable to achieve a 1-log10 kill with the two PHMB-based MPDSs against 10 clinical isolates of F. solani (ISO 1472921 requires a 1.0-log reduction in colony-forming units for the fungal species). Ide et al.16 evaluated and compared the in vitro effectiveness of MPDS to inhibit fungal colonization of FDA (Food and Drug Administration) contact lens groups. They showed that fungi were more likely (43.7 vs. 18.6%, p = 0.062) to be recovered from the PHMB-based product (ReNu MultiPlus, 0.0001% PHMB) than from the alexidine-based product or POLYQUAD-based solution. They conclude that the antifungal activity of contaminated MPDSs may be insufficient to prevent fungal colonization of contact lens materials despite meeting or exceeding the FDA stand-alone test criteria. Colonized lens may increase the risk of fungal keratitis. Loss of disinfecting efficacy after lenses are soaked in PHMB products for various periods of time has been demonstrated in several studies. Loss of disinfection efficacy during the disinfection process appears the most likely initiator of sustained Fusarium contamination of the solution, lens, and case.22 Polyhexamethylene biguanide has been used for the treatment of Acanthamoeba keratitis at concentrations of 0.02 to 0.053% and for the treatment of soft contact lensYrelated Fusarium
TABLE 2.
Comparison of the concentrations of four disinfectants in MPDSs with their MIC90 values against ocular pathogenic F. solani and A. flavus Disinfectant concentrations in MPDSs, Kg/mL 0.00005% PHMB (0.5) 0.0001% PHMB (1) 0.0005% PHMB (5) 0.002% TMR (20) 0.005% TMR (50) 0.00014% CPC (1.4) 0.003% CHD (30) 0.006% CHD (60) 0.015% CHD (150)
MIC90 against F. solani, Kg/mL
Concentration/MIC90 value
MIC90 against A. flavus, Kg/mL
4 4 4 0.0313 0.0313 2 32 32 32
0.125 0.25 1.25 639 1597 0.7 0.9 1.9 4.7
16 16 16 0.0625 0.0625 2 32 32 32
Concentration/MIC90 value
TMR, thimerosal; CPC, cetylpyridinium chloride; CHD, chlorhexidine. Optometry and Vision Science, Vol. 91, No. 12, December 2014
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0.0313 0.0625 0.3125 320 800 0.7 0.9 1.9 4.7
Contact Lens Solution Disinfectants and Filamentous FungiVXu et al.
keratitis at a concentration of 0.02% without causing adverse effects.18,23 When comparing the concentrations of PHMB in eye drops with its concentrations in MPDSs, the concentrations of PHMB in eye drops are 40 to 500 times as high as its concentrations in MPDSs. Based on our experimental results and evidence from the above studies, we propose that PHMB’s concentrations in MPDSs should be appropriately increased within tolerable limits to enhance antimicrobial effects. Thimerosal is a preservative commonly used in ophthalmic solutions, topical medicine, and vaccines. It is also used in MPDSs.24Y27 In this study, MIC90 values of thimerosal were 0.0313 and 0.0625 Kg/mL for ocular pathogenic F. solani and A. flavus, respectively. Thimerosal showed the highest levels of antimicrobial activity against fungal species and proved to be the most effective disinfectant. Our results were consistent with those obtained by us in a former investigation. That study showed that thimerosal’s antifungal activity was significantly superior to those of amphotericin B and natamycin against 244 ocular pathogenic fungi in vitro.28 Our results were also consistent with those from another study in which the MPDS Hydrocare with thimerosal was highly effective in eliminating A. fumigatus (ATCC 36607) contamination.24 In MPDSs, thimerosal is present at 20 or 50 Kg/mL (0.002 or 0.005%).18,20 Based on our data, when comparing the MIC90 values of thimerosal with its concentrations in MPDSs, the concentration of thimerosal is 639 times or 1597 times as high as its MIC90 value against ocular pathogenic F. solani and 320 times or 800 times as high as its MIC90 value against A. flavus (Table 2). Therefore, both ocular pathogenic F. solani and A. flavus were found very susceptible to thimerosal concentrations much lower than those present in MPDS formulations. So far, there is a clinical finding that an adverse effect induced by thimerosal as a preservative in MPDSs is ocular delayed hypersensitivity.29Y31 Patients with delayed hypersensitivity to thimerosal may develop conjunctival hyperemia, corneal infiltrates, and intolerance to lens wear with the use of MPDSs containing thimerosal. Because of allergic reactions, thimerosal is less used today in MPDSs. Based on our experimental results, we propose that thimerosal’s concentrations in MPDSs should be markedly decreased within antimicrobial effects to decrease side effects. Cetylpyridinium chloride is the antiseptic component in the widely used mouthwashes and is also the antiseptic component in some MPDSs.32,33 Although previous studies have documented cetylpyridinium chloride activity versus Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa,33 here our study first reported cetylpyridinium chloride activity versus ocular pathogenic F. solani and A. flavus isolates in vitro. In MPDSs, cetylpyridinium chloride is present at 1.4 Kg/mL (0.00014%).33 Based on our data, when comparing the MIC90 values of cetylpyridinium chloride with its concentration in MCLDSs, the concentration of cetylpyridinium chloride is lower than its MIC90 values against ocular pathogenic F. solani and A. flavus isolates (Table 2). In this study, cetylpyridinium chloride had a much lower MIC90 value than PHMB against A. flavus isolates; it appeared to be more effective than PHMB against ocular pathogenic A. flavus isolates. Chlorhexidine is a commonly used preservative in eye preparations and is also the antiseptic component in some MPDSs.25Y27
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Although previous studies have documented chlorhexidine activity versus S. aureus, E. coli, P. aeruginosa, Candida albicans, and Acanthamoeba,26,27,34 here our study first reported chlorhexidine activity versus ocular pathogenic F. solani and A. flavus isolates in vitro. In MPDSs, chlorhexidine is most commonly present at 30 or 60 Kg/mL (0.003 or 0.006%) and rarely present at 150 Kg/ mL (0.015%).13,25Y27 Based on our data, when comparing the MIC90 values of chlorhexidine with its concentrations in MPDSs, the concentration of chlorhexidine 0.003% is lower than its MIC90 values against F. solani and A. flavus isolates, and the concentration of chlorhexidine 0.006 or 0.015% is 1.9 times or 4.7 times as high as its MIC90 values against F. solani and A. flavus isolates, respectively (Table 2). Our present results indicate that in MPDSs, 0.003% chlorhexidine is a sublethal level, but 0.006 and 0.015% chlorhexidine are overlethal levels for ocular pathogenic F. solani and A. flavus isolates. The experimental protocol used in this study represents a different approach from those taken by ISO 14729 where several sequential steps and a standard strain F. solani ATCC 36031 are used to determine an MPDS’ efficacy. This study’s design focused on the efficacy of disinfectants PHMB, thimerosal, cetylpyridinium chloride, and chlorhexidine against ocular pathogenic F. solani and A. flavus isolates. F. solani is one of the most common causes of fungal keratitis and, therefore, is used as a challenge organism in microbiology disinfectant tests.21 We contributed additional data (MIC90 values) for the previously rare tested ocular pathogenic F. solani and A. flavus isolates. A notable result was that ocular pathogenic F. solani isolates tested in our investigation appeared to be less susceptible to PHMB than the ATCC strain tested in other investigations. Our result was in relative concordance with some studies.1,2,6Y9 In addition, A. flavus has emerged as a predominant ocular fungal pathogen12,35,36 but is not used as a challenge organism in microbiology disinfectant tests. Our studies provided additional evidence to determine the effectiveness of PHMB against A. flavus. In our study, the MIC90 value of PHMB was 16 Kg/mL for A. flavus isolates and A. flavus appeared to be less susceptible than F. solani to the antifungal action of PHMB. Our study, like those of others,6Y9 revealed inadequacies in the testing procedures recommended by ISO 14729, where only one Fusarium isolate is used to challenge the disinfectants.21 The use of a single laboratory strain and a single source of inoculum may provide a false sense of assurance that the disinfecting solution is effective.9 We think that the test could be improved by the use of both laboratory strains and multiple clinical strains to reduce the risk of fungal keratitis associated with contact lens wear. In conclusion, in this study, as a disinfectant used in MPDSs, thimerosal showed the highest levels of antimicrobial activity against ocular pathogenic F. solani and A. flavus isolates. The concentrations of PHMB (0.0001%), cetylpyridinium chloride (0.00014%), and chlorhexidine (0.003%) in MPDSs are sublethal levels for ocular pathogenic F. solani and A. flavus isolates. Although multiple ingredients within MPDSs play a role in antimicrobial efficacy, antimicrobial activity may be significantly influenced by the disinfectants used in the solution formulations.
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1444 Contact Lens Solution Disinfectants and Filamentous FungiVXu et al.
ACKNOWLEDGMENTS This work was supported by the National Natural Science Fund of China (81241033), Fund of Bureau of Health of Henan Province (201002012), and Fund of Bureau of Science and Technology of Henan Province (0641130305). Received March 9, 2014; accepted June 16, 2014.
REFERENCES 1. Levy B, Heiler D, Norton S. Report on testing from an investigation of Fusarium keratitis in contact lens wearers. Eye Contact Lens 2006;32:256Y61. 2. Levy B. Infectious keratitis: what have we learned? Eye Contact Lens 2007;33:418Y20. 3. Chang DC, Grant GB, O’Donnell K, Wannemuehler KA, NobleWang J, Rao CY, Jacobson LM, Crowell CS, Sneed RS, Lewis FM, Schaffzin JK, Kainer MA, Genese CA, Alfonso EC, Jones DB, Srinivasan A, Fridkin SK, Park BJ. Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution. JAMA 2006; 296:953Y63. 4. Donnio A, Van Nuoi DN, Catanese M, Desbois N, Ayeboua L, Merle H. Outbreak of keratomycosis attributable to Fusarium solani in the French West Indies. Am J Ophthalmol 2007;143:356Y8. 5. Khor WB, Aung T, Saw SM, Wong TY, Tambyah PA, Tan AL, Beuerman R, Lim L, Chan WK, Heng WJ, Lim J, Loh RS, Lee SB, Tan DT. An outbreak of Fusarium keratitis associated with contact lens wear in Singapore. JAMA 2006;295:2867Y73. 6. Zhang S, Ahearn DG, Stulting RD, Schwam BL, Simmons RB, Pierce GE, Crow SA, Jr. Differences among strains of the Fusarium oxysporum-F. solani complexes in their penetration of hydrogel contact lenses and subsequent susceptibility to multipurpose contact lens disinfection solutions. Cornea 2007;26:1249Y54. 7. Hume EB, Flanagan J, Masoudi S, Zhu H, Cole N, Willcox MD. Soft contact lens disinfection solution efficacy: clinical Fusarium isolates vs. ATCC 36031. Optom Vis Sci 2009;86:415Y9. 8. Kilvington S, Lam A, Nikolic M, Brady N. Resistance and growth of Fusarium species in contact lens disinfectant solutions. Optom Vis Sci 2013;90:430Y8. 9. Boost M, Lai S, Ma C, Cho P. Do multipurpose contact lens disinfecting solutions work effectively against non-FDA/ISO recommended strains of bacteria and fungi? Ophthalmic Physiol Opt 2010;30:12Y9. 10. Wang DL. Medical MycologyVGuide to Laboratory Examination. Beijing, China: People’s Medical Publishing House; 2005. 11. National Committee for Clinical Laboratory Standards. Reference method for broth dilution antifungal susceptibility testing of filamentous fungi; approved standard. Document M38-A2. Wayne, PA: NCCLS; 2002. 12. Xu Y, Gao C, Li X, He Y, Zhou L, Pang G, Sun S. In vitro antifungal activity of silver nanoparticles against ocular pathogenic filamentous fungi. J Ocul Pharmacol Ther 2013;29:270Y4. 13. Hiti K, Walochnik J, Maria Haller-Schober E, Faschinger C, Aspock H. Efficacy of contact lens storage solutions against different Acanthamoeba strains. Cornea 2006;25:423Y7. 14. Kilvington S, Powell CH, Lam A, Lonnen J. Antimicrobial efficacy of multi-purpose contact lens disinfectant solutions following evaporation. Cont Lens Anterior Eye 2011;34:183Y7.
15. Santodomingo-Rubido J, Mori O, Kawaminami S. Cytotoxicity and antimicrobial activity of six multipurpose soft contact lens disinfecting solutions. Ophthalmic Physiol Opt 2006;26:476Y82. 16. Ide T, Miller D, Alfonso EC, O’Brien TP. Impact of contact lens group on antifungal efficacy of multipurpose disinfecting contact lens solutions. Eye Contact Lens 2008;34:151Y9. 17. Rebong RA, Santaella RM, Goldhagen BE, Majka CP, Perfect JR, Steinbach WJ, Afshari NA. Polyhexamethylene biguanide and calcineurin inhibitors as novel antifungal treatments for Aspergillus keratitis. Invest Ophthalmol Vis Sci 2011;52:7309Y15. 18. Behrens-Baumann W, Seibold M, Hofmuller W, Walter S, Haeberle H, Wecke T, Tammer I, Tintelnot K. Benefit of polyhexamethylene biguanide in Fusarium keratitis. Ophthalmic Res 2012;48:171Y6. 19. Messick CR, Pendland SL, Moshirfar M, Fiscella RG, Losnedahl KJ, Schriever CA, Schreckenberger PC. In-vitro activity of polyhexamethylene biguanide (PHMB) against fungal isolates associated with infective keratitis. J Antimicrob Chemother 1999; 44:297Y8. 20. Vehige JG, Simmons PA, Anger C, Graham R, Tran L, Brady N. Cytoprotective properties of carboxymethyl cellulose (CMC) when used prior to wearing contact lenses treated with cationic disinfecting agents. Eye Contact Lens 2003;29:177Y80. 21. International Organization for Standardization (ISO). Ophthalmic OpticsVContact Lens ProductsVMicrobial Requirements and Test Method for Products and Regimes for Hygienic Management of Contact Lenses: ISO 14729. Geneva, Switzerland: ISO; 2001. 22. Rosenthal RA, Dassanayake NL, Schlitzer RL, Schlech BA, Meadows DL, Stone RP. Biocide uptake in contact lenses and loss of fungicidal activity during storage of contact lenses. Eye Contact Lens 2006;32:262Y6. 23. Duguid IG, Dart JK, Morlet N, Allan BD, Matheson M, Ficker L, Tuft S. Outcome of Acanthamoeba keratitis treated with polyhexamethyl biguanide and propamidine. Ophthalmology 1997; 104:1587Y92. 24. Connor CG, Presley L, Finchum SM, Steel SA. The effectiveness of several current soft contact lens care systems against Aspergillus. CLAO J 1998;24:82Y4. 25. Imayasu M, Moriyama T, Ichijima H, Ohashi J, Petroll WM, Jester JV, Cavanagh HD. The effects of daily wear of rigid gas permeable contact lenses treated with contact lens care solutions containing preservatives on the rabbit cornea. CLAO J 1994; 20:183Y8. 26. Keeven J, Wrobel S, Portoles M, DeCicco BT. Evaluating the preservative effectiveness of RGP lens care solutions. CLAO J 1995; 21:238Y41. 27. Zanetti S, Fiori PL, Pinna A, Usai S, Carta F, Fadda G. Susceptibility of Acanthamoeba castellanii to contact lens disinfecting solutions. Antimicrob Agents Chemother 1995;39:1596Y8. 28. Xu Y, Pang G, Zhao D, Gao C, Zhou L, Sun S, Wang B. In vitro activity of thimerosal against ocular pathogenic fungi. Antimicrob Agents Chemother 2010;54:536Y9. 29. Wilson LA, McNatt J, Reitschel R. Delayed hypersensitivity to thimerosal in soft contact lens wearers. Ophthalmology 1981; 88:804Y9. 30. Mondino BJ, Salamon SM, Zaidman GW. Allergic and toxic reactions of soft contact lens wearers. Surv Ophthalmol 1982; 26:337Y44. 31. Wright P, Mackie I. Preservative-related problems in soft contact lens wearers. Trans Ophthalmol Soc U K 1982;102(Pt. 1):3Y6.
Optometry and Vision Science, Vol. 91, No. 12, December 2014
Copyright © American Academy of Optometry. Unauthorized reproduction of this article is prohibited.
Contact Lens Solution Disinfectants and Filamentous FungiVXu et al. 32. Yu ZJ, Huth SW. Cetylpyridinium chloride as an antimicrobial agent in ophthalmic compositions. US patent 7,578,996. August 25, 2009. 33. Chen JY, He T, Jia XH. [The sorption was observed on the active ingredient of contact lens care solution by germicidal test.]. Chin Med Equip News 2006;2:65Y7. 34. Zhu B, Liu N, Lin LY, Li PB, Yin T. [Study on germicidal efficacy and toxicity of multi-functional contact lens cleaning-disinfection solution.]. Int J Lab Med 2009;30:274Y6. 35. Xu Y, Zhao D, Gao C, Zhou L, Pang G, Sun S. In vitro activity of phenylmercuric acetate against ocular pathogenic fungi. J Antimicrob Chemother 2012;67:1941Y4.
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36. Xie L, Zhai H, Zhao J, Sun S, Shi W, Dong X. Antifungal susceptibility for common pathogens of fungal keratitis in Shandong Province, China. Am J Ophthalmol 2008;146:260Y5.
Yan Xu Laboratory of Ocular Pharmacology Henan Eye Institute and Henan Eye Hospital 7 Weiwu Rd Zhengzhou, 450003 China e-mail: [email protected]
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