Contact lens-related keratitis is a rare, sight-threatening complication affecting approximately four in 10,000 daily wear contact lens wearers and approximately 20 in 10,000 extended-wear contact lens wearers.1,2 Most often, keratitis is microbially mediated, with Pseudomonas and Staphylococcus species the predominating infecting bacteria.3,4 However, up to 35% of reported cases of keratitis are attributed to fungus,5,6 with Fusarium solani the causative agent in 25 to 62% of these.6–8F. solani is a soil and aqueous dwelling plant pathogen. It is not transmitted between humans9; however, it is an opportunistic pathogen that can colonize corneal epithelia after trauma or injury to the eye.10
Until the beginning of 2006, there had been no reports regarding the association of multipurpose disinfection solutions (MPDS) with fungal keratitis.11 Beginning in 2006, increased incidences of Fusarium keratitis were reported among contact lens wearers in Asia12,13 and the United States,9,14–16 stimulating investigation into the possible contamination or inadequate disinfecting efficacy of MPDS.5,17,18 One study observed a seven fold increase in corneal ulceration due to Fusarium from July 2005 to May 2006 compared with the previous 30 months.17 In the later study, ReNu MoistureLoc was identified as the main risk factor for fungal keratitis development resulting in a subsequent recall of the solution.11 ReNu MoistureLoc was sterile and met regulatory guidelines for antimicrobial activity. However, a multivariant interaction of poor hygienic practices, a decline in antifungal efficacy of the solution related to desiccation or dilution, and the absorption or adsorption of antimicrobial components onto/into lens cases or lenses allowed for enhanced growth of the Fusarium, and subsequent infection. Lack of a manual rubbing-cleaning step in the disinfection regimen with this MPDS was further considered a risk factor for keratitis.19
Soft multipurpose contact lens disinfecting solutions contain polyquaternium (polyquad) and/or myristamidopropyldimethylamine (aldox) or polyhexamethylene biguanide (PHMB) as the main antibacterial and antifungal agents.20–22 Even in the presence of such agents, it has been shown that some MPDS are more frequently involved in cases of contact lens-related Fusarium keratitis.11 This might, in part, be attributed to the uptake and release kinetic profile of MPDS, which could ultimately affect their fungicidal activity.10 There has, to-date, been no comprehensive investigation into the fungicidal activity of MPDS against a range of F. solani clinical isolates.
Here, we report the first comprehensive assessment of the disinfecting efficacy of five MPDS (two of which are not commercially available including the recalled ReNu MoistureLoc) on 10 F. solani clinical isolates and the standard ISO ATCC 36031 strain using the manufacturers’ minimum recommended disinfection time (MRDT). Disinfecting efficacy was assessed relative to the ISO stand-alone disinfection test (ISO/CD 14729),23 which requires a 1.0-log reduction in colony forming units for the fungal species.
MATERIALS AND METHODS
Ten clinical isolates of F. solani were used in this study (Fsol 2-Fsol 11) as well as the standard ISO-recommended ATCC 36031 strain. All clinical isolates were obtained from corneal ulcers and were kindly provided by Ailsa Hocking (Food Science Australia, CSIRO, North Ryde, Australia) and David Ellis (Women and Children’s Hospital, Adelaide, Australia). All corneal ulcer isolates were from different patients and strains were identified by microscopy morphology and culture. Isolates were stored at −80°C in 30% glycerol and tryptone soya broth (Oxoid, Basingstoke, UK).
Effect of Filtration on Stand-Alone Disinfection
Representative strains, ISO standard ATCC 36031 and a clinical strain (Fsol 5) were inoculated on potato dextrose agar, grown at 25°C for 10 to 14 days, and then harvested by scraping of the agar surface. To compare the process of filtering the fungal suspension, the suspension was filtered through a 0.7-μm filter to separate conidia from hyphae or left unfiltered. Approximately 1 × 108 CFU/ml were suspended in Dulbecco’s phosphate-buffered saline (pH 7.3) and vortexed for 2 to 3 min. Retrospective plate counts were performed to ensure that 1 × 108 CFU/ml were present in this suspension. Ten microliters of the conidial or mixed condidial or hyphal suspension were added to 0.990 ml, giving a final fungal concentration of approximately 1 × 106 CFU/ml, in one of two representative MPDS (D or E, Table 1) and incubated at 20 to 25°C for MRDT (4 or 6 h, Table 1). After the MRDT, 0.100 ml were taken from each tube and added to 0.900 ml Difco Dey-Engley neutralizing broth (BD Diagnostic Systems, Sparks, MD, U.S.) and incubated for 10 to 15 min (20 to 25°C) to halt disinfection. Serial dilutions were performed in neutralizing media, and plated out (0.100 ml) in duplicate. Plates were incubated for 2 to 3 days and colonies formed were enumerated. All plates exhibiting no colonies were kept for 14 days more to confirm absence of growth. Experiments were performed in triplicate four times and average log reduction for each MPDS/strain combination was calculated.
For the ATCC 36031 strain and 10 clinical isolates (Fsol 2–11), fungi were grown as above and scrapes were collected. As a mixed conidia and hyphae suspension mimics a more “real life” situation (such as growth in cases and on lenses), we chose to test the MPDS against a mixed suspension of both conidia and hyphae in which the suspension was not filtered and placed directly into MPDS. Fungal suspensions were inoculated into one of five MPDS (Table 1), incubated for the MRDT, and processed as described earlier. Experiments were performed in triplicate four times and average log reductions for each MPDS/strain combination were calculated.
Differences between filtered and unfiltered suspensions for the ATCC strain and variations in the disinfection ability of the various MPDS for each of the 11 strains were determined using a paired two-tailed Student’s t-test. Linear mixed models were used to determine differences between sample type, solution groups, and their interaction. Posthoc comparisons were adjusted using Bonferroni correction. Level of significance was set at 5%.
Two solutions were tested on two Fusarium strains to assess the effect of filtering to remove hyphae. For the ISO strain, there was no difference between unfiltered and filtered fungal suspensions on their ability to resist disinfection by MPDS (Fig. 1A). The clinical isolate (Fsol 5) was more resistant to disinfection in each of solutions D and E compared with the ISO strain (Fig. 1). Solution D showed greater disinfecting efficacy than solution E for both the ISO strain and the clinical isolate. There was a significant difference between filtered vs. unfiltered preparations of the clinical isolate for solution D (Fig. 1B) with the unfiltered preparation proving significantly more resistant (p < 0.05). There was no difference in filtered vs. unfiltered for solution E.
Results for MPDS solutions tested against each isolate are presented in Table 2. All except solution E met the stand-alone criteria of 1-log reduction against the ISO-recommended ATCC 36031strain (Table 2). The ATCC strain was significantly more susceptible to MPDS disinfection than the clinical isolates (p < 0.05). There was wide variation in the disinfecting efficacy of MPDS tested against each clinical isolate. However, the efficacy of each solution relative to each isolate was similar (Table 2). None of the solutions tested met the stand-alone criteria for all clinical isolates examined. Furthermore, two clinical isolates (Fsol 3 and Fsol 5) were resistant to disinfection by all MPDS tested (Table 2).
The polyquaternium-based disinfection solutions with either aldox or alexidine proved more effective overall than the PHMB-based MPDS against all strains of F. solani examined. Posthoc comparison of solutions on clinical isolates showed no significant difference between solutions A and E. Further, there was no significant difference in the disinfection efficacy between solutions B, C, and D for all clinical isolates but these three solutions demonstrated significantly better disinfection efficacy compared with solutions A and E (p < 0.001). Greatest log reductions were seen for all isolates with solution B. Solutions C and D performed similarly to each other (Table 2).
The primary criterion of the ISO 14729 stand-alone test in relation to fungicidal efficacy is an average reduction greater than or equal to 1.0 log in CFU within the MRDT.23 To date, challenge organisms have been laboratory strains. It is now widely accepted that laboratory strain organisms are often less virulent than clinical isolates. This lack of virulence in laboratory strains is due to generations of laboratory cultivation without competition, which results in a loss of virulence factors. Concomitantly clinical pathogens have acquired increased resistance because of competition in face of attempted eradication outside the laboratory.24–26 Thus, it is important, in light of the recently reported increased incidence of Fusarium keratitis in contact lens wearers, to determine the efficacy of MPDS against a range of clinical ocular F. solani isolates. We were also interested in testing, in a controlled environment, the efficacy of two contact lens solutions no longer commercially available (Solutions B and E), withdrawn from the market because of their association with an increased incidence rate of Fusarium and Acanthamoeba keratitis.27
Compared with filtered suspensions, unfiltered fungal suspensions used in the present study exhibited a slightly increased resistance to the MPDS tested. The increased resistance reached significance for the clinical isolate with solution D. Use of this unfiltered suspension in an attempt to model a more realistic use of MPDS against F. solani may account for the increased resistance exhibited. An unfiltered clinical isolate suspension more closely resembles the situation that would be found outside the laboratory and thus should be considered as a more informative test of efficacy for clinical applications.
Predictably, almost all solutions tested showed greater log reductions with the ATCC strain than the clinical strains. The ISO recommended F. solani strain met the 1 log reduction criteria for all but one MPDS tested. Levy et al.18 reported similar results; however, the magnitude of reduction in CFU was lower in the present study than that reported previously.18 We determined that if unfiltered fungal suspensions are used, an increase in resistance to the MPDS can occur, which may explain in part the differences between ours and previous findings and the failure of one of the solutions to pass the 1 log reduction criteria.
Two of the clinical isolates (Fsol 3 and Fsol 5) were not susceptible to disinfection by any of the MPDS and, indeed, almost half of the clinical isolates tested did not meet the ISO disinfection criteria for most of the solutions tested. The solutions that were least efficacious were solutions A and E. Solutions A and E are formulated with different disinfecting agents than solutions B, C, and D.
Solutions A and E are formulated with PHMB, solutions C and D with polyquartenium-1 (polyquad) and myristamidopropyldimethylamine (aldox), and solution B with alexidine and polyquartenium-10 as disinfecting agents. The activity of PHMB is dependent on solution formulation.22 Cellulose-based agents rapidly bind to PHMB reducing its chemical availability and bioactivity,28 whereas sodium and potassium chloride reduce the cationic active sites and thus cytotoxicity.22 Solution E contains hydroxypropyl methylcellulose as well as both sodium chloride and potassium chloride all of which contribute to the reduction in cytoxicity of PHMB. Solution A contains sodium chloride. The aldox in solutions C and D is most likely to be the main component contributing to the disinfecting efficacy of these solutions as it has previously been shown to be more active than polyquad for fungi.20 The disinfecting efficacy exhibited by solution B might be due, in part, to alexidine or the combination of alexidine and polyquaternium-10. Ironically, because of an increased association with Fusarium keratitis, solution B has previously been recalled. Solution B as well as other and other MPDS tested subsequent to the recall of ReNu Moistureloc have shown reduced fungicidal activity against group IV lenses inoculated with Fusarium.10 Additionally, other researchers have shown that reuse of solution B for more than 2 days in which worn contact lenses are stored in contact lens cases filled with the same MPDS as the previous day, with or without topping off with fresh solution, results in loss of activity against Fusarium.18 That one clinical isolate showed similar susceptibility to the ISO-recommended ATCC strain in the stand-alone test but was found to be more robust than the ATCC strain on desiccation in contact lens solution is further evidence supporting the need for more rigorous testing of solutions than the current ISO biocidal test demands.18 Additional evidence supporting the need for more complex test conditions is found in a separate study in which solution B, but not other MPDS, lost much of its antifungal disinfecting activity under complex stress conditions, resulting in Fusarium colonization of contact lens cases and MPDS containers.29 This same study showed that clinical isolates of Fusarium are better able to penetrate contact lenses and survive MPDS disinfection than the ISO standard strain further supporting the need for more rigorous testing.
Because almost half the clinical isolates were recalcitrant to disinfection by the majority of MPDS, it would seem that combinations of presently available fungicides will still prove inadequate for a large number of clinical F. solani isolates.
Lastly, it must be noted that the ISO standard test is performed under well-controlled laboratory conditions while in practice, the use of MPDS has been associated with contact lens user non-compliance/malpractice increasing the likelihood of a combination of the presence of organic soil, wearing of used contact lenses or bacterial biofilms that would further reduce disinfecting efficacy of MPDS.30–32 Thus, the current practice of the stand-alone test run under well-controlled conditions and using a laboratory strain that is known to be less pathogenic than many clinical isolates would be expected to result in unrealistically optimistic performance outcomes. In view of the results of this study, the use of clinical isolates should be encouraged in the practice of the ISO stand-alone test. However, future studies assessing the disinfecting efficacy of MPDS should also consider using both laboratory strains and clinical isolates as well as undertaking testing with clean and worn contact lenses, the presence of soil and simulating solution evaporation.
The authors would like to thank Debbie McDonald for assistance in formatting this manuscript.
Institute for Eye Research
The University of New South Wales
Level 3, Rupert Myers Building
Sydney, NSW 2052
1. Poggio EC, Glynn RJ, Schein OD, Seddon JM, Shannon MJ, Scardino VA, Kenyon KR. The incidence of ulcerative keratitis among users of daily-wear and extended-wear soft contact lenses. N Engl J Med 1989;321:779–83.
2. Cheng KH, Leung SL, Hoekman HW, Beekhuis WH, Mulder PG, Geerards AJ, Kijlstra A. Incidence of contact-lens-associated microbial keratitis and its related morbidity. Lancet 1999;354:181–5.
3. Chalita MR, Hofling-Lima AL, Paranhos A Jr, Schor P, Belfort R Jr. Shifting trends in in vitro antibiotic susceptibilities for common ocular isolates during a period of 15 years. Am J Ophthalmol 2004;137:43–51.
4. Pachigolla G, Blomquist P, Cavanagh HD. Microbial keratitis pathogens and antibiotic susceptibilities: a 5-year review of cases at an urban county hospital in north Texas. Eye Contact Lens
5. Alfonso EC, Miller D, Cantu-Dibildox J, O’Brien TP, Schein OD. Fungal keratitis
associated with non-therapeutic soft contact lenses. Am J Ophthalmol 2006;142:154–5.
6. Liesegang TJ, Forster RK. Spectrum of microbial keratitis in South Florida. Am J Ophthalmol 1980;90:38–47.
7. Rosa RH Jr, Miller D, Alfonso EC. The changing spectrum of fungal keratitis
in south Florida. Ophthalmology 1994;101:1005–13.
8. Tanure MA, Cohen EJ, Sudesh S, Rapuano CJ, Laibson PR. Spectrum of fungal keratitis
at Wills Eye Hospital, Philadelphia, Pennsylvania. Cornea 2000;19:307–12.
9. Centers for Disease Control and Prevention (CDC). Fusarium
keratitis— multiple states, 2006. MMWR Morb Mortal Wkly Rep 2006;55:400–1.
10. 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
11. Dyavaiah M, Ramani R, Chu DS, Ritterband DC, Shah MK, Samsonoff WA, Chaturvedi S, Chaturvedi V. Molecular characterization, biofilm analysis and experimental biofouling study of Fusarium
isolates from recent cases of fungal keratitis
in New York State. BMC Ophthalmol 2007;7:1–9.
12. Schein OD, Glynn RJ, Poggio EC, Seddon JM, Kenyon KR. The relative risk of ulcerative keratitis among users of daily-wear and extended-wear soft contact lenses. A case-control study. Microbial Keratitis Study Group. N Engl J Med 1989;321:773–8.
13. 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;295:2867–73.
14. Alfonso EC, Cantu-Dibildox J, Munir WM, Miller D, O’Brien TP, Karp CL, Yoo SH, Forster RK, Culbertson WW, Donaldson K, Rodila J, Lee Y. Insurgence of Fusarium
keratitis associated with contact lens
wear. Arch Ophthalmol 2006;124:941–7.
15. Bernal MD, Acharya NR, Lietman TM, Strauss EC, McLeod SD, Hwang DG. Outbreak of Fusarium
keratitis in soft contact lens
wearers in San Francisco. Arch Ophthalmol 2006;124:1051–3.
16. Chang DC, Grant GB, O’Donnell K, Wannemuehler KA, Noble-Wang 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;296:953–63.
17. Gorscak JJ, Ayres BD, Bhagat N, Hammersmith KM, Rapuano CJ, Cohen EJ, Burday M, Mirani N, Jungkind D, Chu DS. An outbreak of Fusarium
keratitis associated with contact lens
use in the northeastern United States. Cornea 2007;26:1187–94.
18. Levy B, Heiler D, Norton S. Report on testing from an investigation of fusarium keratitis in contact lens
wearers. Eye Contact Lens
19. Ahearn DG, Zhang S, Stulting RD, Schwam BL, Simmons RB, Ward MA, Pierce GE, Crow SA Jr. Fusarium
keratitis and contact lens
wear: facts and speculations. Med Mycol 2008;46:397–410.
20. Codling CE, Maillard JY, Russell AD. Aspects of the antimicrobial mechanisms of action of a polyquaternium and an amidoamine. J Antimicrob Chemother 2003;51:1153–8.
21. McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev 1999;12:147–79.
22. Santodomingo-Rubido J, Mori O, Kawaminami S. Cytotoxicity and antimicrobial activity of six multipurpose soft contact lens
disinfecting solutions. Ophthalmic Physiol Opt 2006;26:476–82.
23. Rosenthal RA, Sutton SV, Schlech BA. Review of standard for evaluating the effectiveness of contact lens
disinfectants. PDA J Pharm Sci Technol 2002;56:37–50.
24. Cooper VS, Lenski RE. The population genetics of ecological specialization in evolving Escherichia coli
populations. Nature 2000;407:736–9.
25. Fux CA, Shirtliff M, Stoodley P, Costerton JW. Can laboratory reference strains mirror “real-world” pathogenesis? Trends Microbiol 2005;13:58–63.
26. Riley MS, Cooper VS, Lenski RE, Forney LJ, Marsh TL. Rapid phenotypic change and diversification of a soil bacterium during 1000 generations of experimental evolution. Microbiology 2001;147:995–1006.
27. Patel A, Hammersmith K. Contact lens
-related microbial keratitis: recent outbreaks. Curr Opin Ophthalmol 2008;19:302–6.
28. 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
29. Zhang S, Ahearn DG, Noble-Wang JA, Stulting RD, Schwam BL, Simmons RB, Pierce GE, Crow SA Jr. Growth and survival of Fusarium solani
complex on stressed multipurpose contact lens
care solution films on plastic surfaces in situ and in vitro. Cornea 2006;25:1210–6.
30. Dudley R, Matin A, Alsam S, Sissons J, Maghsood AH, Khan NA. Acanthamoeba isolates belonging to T1, T2, T3, T4 but not T7 encyst in response to increased osmolarity and cysts do not bind to human corneal epithelial cells. Acta Trop 2005;95:100–8.
31. Pens CJ, da Costa M, Fadanelli C, Caumo K, Rott M. Acanthamoeba
spp. and bacterial contamination in contact lens
storage cases and the relationship to user profiles. Parasitol Res 2008;103:1241–5.
32. Garate M, Marchant J, Cubillos I, Cao Z, Khan NA, Panjwani N. In vitro pathogenicity of Acanthamoeba
is associated with the expression of the mannose-binding protein. Invest Ophthalmol Vis Sci 2006;47:1056–62.