British Journal of Ophthalmology 1995; 79: 601-605

Acanthamoeba, bacterial, and fungalcontamination of contact lens storage cases

Trevor B Gray, Ray T M Cursons, Jane F Sherwan, Paul R Rose

AbstractBackground-Microbial corneal infectionis the most serious complication of con-tact lens wear. Contact lens cases are arecognised potential source of pathogensassociated with corneal ulcers.Methods-This survey established theincidence of protozoal, bacterial, andfungal contact lens case contamination in101 asymptomatic daily wear cosmeticcontact lens wearers from a domiciliarycontact lens practice.Results-Eighty two (81%) contact lenscases were found to be contaminated, with19 (19%) sterile. Of all contact lens cases,78 (77%) grew bacteria, 24 (24%) fungi,and 20 (20%) protozoa. Acanthamoebaspp were isolated from eight (8%) contactlens cases. Fifty six (55°/0) contact lenscases yielded mixed bacterial contamina-tion. This is the first contact lens casesurvey in which hydrogen peroxide disin-fection was the major method of contactlens disinfection (75°/0 of subjects) and nohome made saline was used. All the con-taminating organisms were shown topossess the enzyme catalase that breaksdown hydrogen peroxide to oxygen andwater. The polymicrobial nature of thebiofihms found in many contact lens casesis illustrated electron micrographically.Conclusion-Based on data from this andprevious studies, the authors concludewith recommendations for contact lenswearers: (1) regular scrubbing of contactlens case interior to disrupt biofilms; (2)exposure of contact lens case to very hotwater (>'70'C) will kill Acanthamoebacontaminants; (3) allow contact lens caseto air dry between uses; (4) if hydrogenperoxide disinfection is preferred, use atwo step system; (5) replace contact lenscase regularly.(BrJ7 Ophthalmol 1995; 79: 601-605)

Contact lens wear is now the most prevalentrisk factor for new cases of corneal ulcersat Moorfields Eye Hospital.' AlthoughPseudomonas aeruginosa is the most importantmicrobial cause of contact lens associated ker-atitis, other bacteria, fungi, or Acanthamoebamay also cause this condition.' 2 To whatextent lens hygiene systems contribute to thiscondition has been the subject of debate.36Although there have been many documentedcases of identical bacterial strains being iso-lated from an infected cornea and the contactlens case used before infection4 7 8 recent evi-dence regarding extended wear contact lenses

suggests that there may be no correlationbetween contact lens hygiene and bacterialkeratitis.1 Insufficient data exist for acan-thamoebae to draw a similar conclusion. It hasbeen suggested that co-contamination of con-tact lens care systems with acanthamoebae andbacterial species capable of supportingamoebic growth may be the first step in thepathogenesis of Acanthamoeba keratitis by theprovision of large inocula of amoebae.9 Werecently described a case of Acanthamoebakeratitis in a disposable lens wearer in which alack of lens hygiene and co-contamination ofthe contact lens case with both bacteria andacanthamoebae was noted.'0 Because contactlenses may be an important vehicle for thetransfer of amoebae from contaminated con-tact lens cases to the cornea, we undertook asurvey of the microbiological quality of thecontact lens cases from asymptomatic dailywear cosmetic contact lens wearers. Particularnote was made of the lens hygiene systemsbeing employed and their relative effectivenessin preventing or inhibiting microbial colonisa-tion of the contact lens case.

Subjects and methods

SUBJECTSOne hundred and one consecutive contactlens wearing clients attending a domiciliaryoptometry practice were recruited. They hadeach used contact lenses for more than 6months, had no eye disease, and were cosmeticcontact lens wearers. Before their annualcheckup visit, each client was offered a freenew contact lens case in exchange for present-ing their old one. They were only informed ofthe contamination survey at the contact lenscase exchange. Most of the recruitmentoccurred during the winter months.The following information was recorded:

client age, sex, occupation, length of contactlens use, contact lens material, type of wear(daily, extended, or occasional), disinfectant,saline, and cleaner. The clients were askedto describe their contact lens disinfectionand cleaning routine, their compliance withproprietary recommendations was assessedas complete compliance, minor faults (forexample, no hand washing), or major faults(for example, no disinfectant used). Presenceof clinical symptoms (for example, redness,irritation) and signs (for example, conjunctivalhyperaemia) was documented. No accuratedetermination of the period of time that thecontact lens case had been in use could bemade because few clients could rememberaccurately.

Department ofOphthalmology,Auckland Hospital,Auckland, NewZealandT B Gray

Department ofPathology, WaikatoHospital, Hamilton,New ZealandR T M CursonsJ F Sherwan

PO Box 9111,Hamilton, NewZealandP R Rose

Correspondence to:Dr T B Gray, Department ofOphthalmology, AucklandHospital, P Bag 92024,Auckland 1, New Zealand.Accepted for publication9 January 1995

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LABORATORY METHODSContact lens cases were coded and batchedbefore being sent to the laboratory where theywere opened under aseptic conditions.Laboratory staff had no access to the clinicaldata detailed above. Any fluid found remainingin the case was discarded. A sterile, unpre-served saline moistened cotton wool swab wasrubbed over all internal surfaces and the swabtip placed in a sterile bijou container. A total of4 ml sterile saline was added to the contact lenscase and then shaken vigorously. This solutionwas transferred to the bijou containing theswab tip. The bijou contents were mixed usinga vortex for 10 seconds, and divided forbacterial, fungal, and amoebal studies.

BACTERIAL ISOLATIONUsing a standard 5 ,ul bacterial loop, the lenscase solution was cultured onto 5% Columbiablood agar and MacConkey agar plates andincubated in air at 30°C for 3 days. After incu-bation all lactose fermenting and non-lactosefermenting Gram negative bacilli were identi-fied to the genus level using a series of manualbiochemical tests." These tests included; oxi-dase, motility, oxidation/fermentation, citrate,methyl red, indole, Voges-Proskauer, andgrowth on MacConkey agar. Catalase wasassayed qualitatively using hydrogen peroxideas outlined in the Manual of ClinicalMicrobiology.'1 Gram positive bacteria wereidentified by their characteristic colonialmorphology and Gram stain appearance. Noattempt was made to further speciate bacteria.

FUNGAL ISOLATIONA 05 ml aliquot of the lens case solution wascultured onto an SAC slope (Sabourauddextrose agar+chloramphenicol 0. 1%+gen-tamicin 0'4%) and incubated in air at 27°C.Cultures were incubated for 14 days beforebeing discarded as negative. Positive cultureswere identified by microscopic and macro-scopic morphology.

AMOEBAL ISOLATIONA 1 0 ml aliquot of the contact lens case solu-tion (as prepared above) was cultured on aPages amoeba saline (PAS) agar plate whichhad previously been spread with a lawn of heatkilled (65°C/30 minutes) Escherichia coli.1I Theplates were incubated in a humidified chamberat 30°C for up to 7 days, and examined every48 hours. Cultured amoebae were identified as

Table 1 Contact ens case disinfectants used by clients in the survey

Product No Active ingredient

Aosept (Ciba Vision) 70 Hydrogen peroxide ('one step')Oxysept 1 and 2 (Allergan) 6 Hydrogen peroxide ('two step')Soaclens (Alcon) 12 ThiomersalBausch & Lomb Multi-purpose solution 3 Polyaminopropyl biguanide 0.00005%Optifree (Alcon) 2 PolyquadBoston Lens 2 ChlorhexidineClean-n-Soak (Allergan) 1 Phenylmercuric nitrate+propylparabensBoil-n-Soak 1 ThiomersalDuraclean (Allergan) 1 Anionic amphoteric surfactantsHydrocare (Allergan) 1 Thiomersal

Table 2 Contact lens cleaning solutions used by clients inthe survey

Product No

Polyclens II (Alcon) 40Miraflow (Ciba Vision) 36Softmate (Bames Hind) 5L.C.65 (Allergan) 5Sorbiclean (Allergan) 4Soft Mate (Barnes Hind) 2Bausch & Lomb Multi-purpose solution 2Oxyclean (Allergan) 1Pliagel (Alcon) 1Boston Lens 1

either Acanthamoeba or other free living amoe-bae by morphology of cyst, flagellate, andtrophozoite stages.

STATISTICAL ANALYSISTreatment groups were compared by x2 analy-sis using Fisher's exact test for significancewhere cell size was small.

ResultsA total of 101 clients (75 female and 26 male)participated. The median age was 32 (range12-65) years, with the median length of con-tact lens use being 6-0 (range 0 5-29) years.Eighty six (85%) patients wore soft contactlenses while the remainder wore hard lenses.Three patients used disposable soft contactlenses (each used a daily wear cleaning and dis-infection regimen with monthly lens disposal).There was no extended wear use of soft contactlenses.

CONTACT LENS HYGIENENinety nine (98%) clients used proprietary dis-infecting solutions (see Table 1) while two(2%) clients used no disinfecting solution.Ninety eight (97%) patients used proprietarycontact lens cleaning solutions (see Table 2),two clients used no contact lens cleaner, andone was unrecorded. Full compliance withrecommended disinfection and cleaning prac-tice was recorded in 61%, minor complianceerrors (for example, no hand washing) in 28%,and major compliance faults (for example, nodisinfection/cleaner) in 1 1%. No patient usedhomemade saline. No heat disinfection unitswere used. There were no overt clinical infec-tions in any of the patients, though many ofthe contact lens cases were observed to haveobvious biofilms (see Fig 1).

MICROBIAL CONTAMINATIONThe results show a significant degree of micro-bial contamination in the contact lens casespresented for analysis. Eighty two (81%) werecontaminated with microbes, leaving only 19(19%) sterile. Twenty four (24%) contact lenscases were colonised by fungi and 20 (20%) byprotozoa. Those recorded as having majorcompliance faults did not have significantlyhigher rates of contact lens case contaminationthan those who described full compliance withrecommended contact lens hygiene. There wasno significant difference in contact lens case

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Table 4 Level of microbial contamination seen withperoxide and non-peroxide disinfection methods (22protozoal contaminants were culturedfrom 20 contact lenscases)

Hydrogen peroxide Non-peroxideContaminant disinfection (%) disinfection (%)

No contamination 11/76 (14) 9/23 (39)Multiple bacterial spp 33/76 (43) 5/23 (22)Fungi 21/76 (28) 3/23 (13)Protozoa:

All spp 18/76 (24) 4/23 (17)Acanthamoeba spp 5/76 (7) 3/23 (13)

*v. s s Y m * R t

Figure 1 Scanning electron micrograph demonstrating microbial biofilm formation(arrowhead) in a used contact lens case, individual bacteria (arrow) are also seen(X3115).

contamination rates or degree of contamina-tion between male and female contact lenswearers.

BACTERIAL CONTAMINATIONSeventy eight (77%) contact lens cases grewbacteria (see Table 3). Notable by theirabsence were Staphylococcus epidermidis andStaphylococcus aureus. Most (56/78) (72%) ofthe culture positive contaminated contact lenscases had mixed bacterial contaminants.

]FUNGAL CONTAMINATIONThere were 24 fungal contaminants, themajority growing Cladosporium spp (10/24) orCandida spp (9/24). Other fungi isolated wereFusarium solani, Aspergillus versicolor,Exophiala spp, and Phoma sp. Most fungi wereisolated in association with bacteria but onthree occasions, fungi were the only microbesisolated.

PROTOZOAL CONTAMINATIONThere were 22 protozoan contaminantsisolated from 20 containers, eight beingAcanthamoeba spp, four Naegleria spp, fourVahlkampfia spp, two Hartmannella spp, threeflagellates, and one ciliate. All protozoa wereisolated in conjunction with bacteria and eighthad fungal co-contamination.

Table 3 Bacteria culturedfrom contact lens cases (totalnumber ofpositive cultures was 78)

Culture Culturepositive positive

Non-fermentativeGram negative: Coliforms:Pseudomonas spp 47/78 Serratia spp 19/8Xanthomonas spp 2/78 Kkebsiella 2/78

Citrobacter 1/78

Other Gram negative: Gram positive:Alcaligenes spp 20/78 Diphtheroids 9/78Acinetobacter spp 11/78 Micrococcus 1/8

Bacillus 1/78

DISINFECTANTSSeventy six (75°/O) clients used hydrogen per-oxide disinfectants and 23 (23%) clients usedcold non-peroxide chemical disinfectionsystems (see Table 1). Relative numbers oforganisms isolated with the different systemsare given in Table 3. There was no significantdifference between the two hydrogen peroxidesystems used in the degree of disinfectionattained.Two clients used no disinfection system. In

neither case were the contact lens cases grosslycontaminated nor were Acanthamoeba spp orfungi cultured. Each grew a single Gram nega-tive isolate (Alcaligenes sp, Acinetobacter sp).

Statistical analysis demonstrated that, withthe exception of Acanthamoeba spp, the non-peroxide systems were associated with lessmicrobial contamination (p<0.05).

DiscussionCorneal infection is the most common visionthreatening complication of contact lens wear.Organisms isolated from contact lens associ-ated corneal ulcers have often been shown tobe identical to those isolated from the contactlens case,4 7 8 10 making the contaminated con-tact lens case a possible replenishable source ofpathogenic microbes. In spite of the apparentadherence to recommended cleaning and dis-infecting regimes, a significant degree ofmicrobial contamination of contact lens caseswas found in this study. Many of the contami-nants identified were potential pathogens andas such should have been prevented by the dis-infectant regimen used.No single reason could be ascertained for

the failure of the contact lens care systemsalthough many contact lens cases wereobserved to contain obvious biofilms (Fig 1).The adherence of microbial biofilms onto plas-tic surfaces and their relative resistance to anti-biotics and antiseptics has been reported.5 6 12These biofilms may have been responsible fordisinfectant failure by providing a continuousseed inoculum.

All microbial contaminants (amoebae, bac-teria, and fungi) isolated from the contact lenscases contained the enzyme catalase. Thisenzyme breaks down hydrogen peroxide towater and oxygen. Continued long term use ofhydrogen peroxide may have selected anaturally resistant population of microbesadapted to survive repeated exposure to 3%hydrogen peroxide. Organisms existing in theoutermost layers of a biofilm are susceptible tobiocides and may release their intracellular

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contents (including catalase) on disruption oftheir cell membrane by hydrogen peroxide.This released catalase may neutralise localhydrogen peroxide activity, protecting otherorganisms living deeper within the biofilm.This suggestion requires further investigation.The 81% incidence of contact lens case

microbial contamination found in this study ishigher than rates of contamination found inother similar studies: 42% in southwestEngland,'3 46% in California,5 and 53% inScotland.'4 This difference may relate to theage of contact lens cases (affording more timeto develop biofilms) in our study, climatic andenvironmental factors, as well as differences inmicrobial culture techniques. Unlike thesesurveys, this study included fungal culture,revealing a 24/101 (24%) contamination rate.Other surveys have reported fungal contamina-tion rates of7% and 9% respectively.3 15The most common microbial contaminant

isolated in descending prevalence were bacte-ria (78%)>fungi (24%)>protozoa (20%).Nearly all contact lens cases had mixed micro-bial populations. The most common bacterialcontaminants isolated were non-fermentativeGram negatives followed by coliforms, otherGram negative, and Gram positive organisms.

This study confirms that Acanthamoeba con-tamination of contact lens cases is far morecommon than Acanthamoeba keratitis. The 8%incidence of contact lens case contaminationwith Acanthamoeba in this study compareswith the 7% in southwest England'3 and 3%(6/178) in Scotland.14

Bacterial co-contamination of contact lenscases growing Acanthamoeba is a commonfeature of this and other contact lens casecontamination studies.'3 14 Acanthamoebaorganisms display selective bacterial feeding,particularly non-fermentative Gram negativesand coliforms.9 It appears that not all non-fermentative Gram negatives are equallysupportive of acanthamoebae. Pseudomonasaeruginosa (the commonest bacterial cause ofmicrobial keratitis) has been shown to producea toxin which is highly lethal to Acanthamoebacastellanii and Acanthamoeba polyphaga, thusthese two eye pathogens may be selectivelyexclusive. 16Three per cent hydrogen peroxide disinfec-

tion systems were the most commonly usedsystem in this study (75% of contact lenscases). It is interesting that change from otherchemical disinfectants to hydrogen peroxidehas been widely recommended with the beliefthat hydrogen peroxide provides greater pro-tection.5 This is the first contact lens case con-tamination survey in which the majority (75%)of contact lens wearers used hydrogen per-oxide disinfection and none used home-madesaline (a reputed major source of contact lenscase contamination). While exposure for 2hours or more to 3% hydrogen peroxide willkill Acanthamoeba cysts and trophozoites, the'one-step' system (used by 70% of the studiedsubjects) employs a catalyst to neutralise thehydrogen peroxide. In vitro study of 'one-step'systems has shown that the exposure to the 3%hydrogen peroxide is too brief to effectively kill

Acanthamoeba cysts.'7 If hydrogen peroxidedisinfection is preferred, the authors recom-mend a two step system in which a properlytimed chemical neutralising agent allowsamoebicidal (and bactericidal) exposure tohydrogen peroxide.

This survey highlights that there is a need forimprovement in contact lens case hygiene.Current contact lens disinfection methods donot appear to be providing a desirable level ofmicrobial protection.

Recommendations to contact lenswearersFrom this and previous data, the authorssuggest the following measures should result inless contact lens case and contact lens con-tamination; thereby possibly reducing the riskof microbial keratitis.

(1) To disrupt accumulated disinfectant-resistant microbial biofilms from internalsurfaces of the contact lens case, regularly scruball internal surfaces with a clean cotton budmoistened with contact lens cleaner.

(2) Regular (for example, fortnightly/monthly) contact lens case heat disinfection byplacing open contact lens case into a container(for example, mug) and filling the containerwith very hot water (1 minute of exposure to> 700C moist heat will kill all Acanthamoebacysts and trophozoites'8). First establish thatthe contact lens case material can withstandhot water exposure.

(3) Leave contact lens case open to air dry afterheat disinfection.

(4) If hydrogen peroxide disinfection is thepreferred regime, use a two step hydrogen perox-ide system.

(5) Of course: wash hands before handlingcontact lens cases; homemade saline never tobe used.

(6) Replace the contact lens case regularly.The use of 'disposable contact lens cases' islikely to make points (1) and (2) less import-ant.The authors thank the Waikato Medical Research Foundationfor their financial support. Some of the replacement contactlens cases were kindly donated by Alcon NZ.

1 Dart JKG, Stapleton F, Minassian D. Contact lenses andother risk factors in microbial keratitis. Lancet 1991; 338:650-3.

2 Dart JKG, Seal DV. Pathogenesis and therapy ofPseudomonas aeruginosa keratitis. Eye 1988; 2: 546-55.

3 Donzis PB, Mondino BJ, Weissman BA, Bruckner DA.Microbiol contamination of contact lens care systems. AmJ Ophthalmol 1987; 104: 325-33.

4 Mayo MS, Schlitzer RL, Ward MA, Wilson LA, AhearnDG. Association of Pseudomonas and Serratia cornealulcers with use of contaminated solutions. Jf Clin Microbiol1987; 25: 1398-400.

5 Wilson LA, Sawant AD, Simmons RB, Aheam DG.Microbial contamination of contact lens storage cases andsolutions. Am Jf Ophthalmol 1990; 110: 193-8.

6 Wilson LA, Sawant AD, Aheam DG. Comparative efficaciesof soft contact lens disinfectant solutions against microbialfilms in lens cases. Arch Ophthalmol 1991; 109: 1115-57.

7 Chalupa E, Swarbrick HA, Holden BA, Sjostrand J. Severecorneal infections associated with contact lens wear.Ophthalmology 1987; 94: 17-22.

8 Mayo MS, Cook WL, Schlitzer RL, Ward MA, Wilson LA,Ahearn DG. Antibiograms, serotypes, and plasmid pro-files of Pseudomonas aenAginosa associated with cornealulcers and contact lens wear. J Clin Microbiol 1986; 24:372-6.

9 Bottone EJ, Madayag RM, Qureshi MN. Acanthamoebakeratitis. Synergy between amoebic and bacterial co-con-taminants in contact lens care systems as a prelude toinfection.

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11 Hendrickson DA, Krenz MM. Reagents and stains. In:Balows A, Hausler WJ, Herrnann KL, Isenberg HD,Shadomy HJ, eds. Manual of clinical microbiology. 5th ed.Washington DC: American Society for Microbiology,1991; chapter 122: 1285-314.

12 Anwar H, Strap JL, Costerton JW. Establishment of agingbiofilms: possible mechanism of bacterial resistance toantimicrobial therapy. Antimicrob Agents Chemother 1992;36: 1347-51.

13 Larkin DF, Kilvington S, Easty DL. Contamination of con-tact lens storage cases by Acanthamoeba and bacteria. BrJOphthalmol 1990; 74: 133-5.

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Microbial contamination of contact lens cases in the westof Scotland. BrjOphthalmol 1993; 77: 41-5.

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16 Qureshi MN, Perez AA, Madayag RM, Bottone EJ.Inhibition ofAcanthamoeba species by Pseudomonas aerug-inosa: rationale for their selective exclusions in comealulcers and contact lens care systems. J Clin Microbiol1993; 31: 1908-10.

17 Silvarny RE, Dougherty JM, McCulley JP, Wood TS,Bowman RW. The effect of currently available contact lensdisinfection systems on Acanthamoeba castellanii andAcanthamoeba polyphaga. Ophthalmology 1990; 97: 286-90.

18 Kilvington S. Moist heat disinfection ofAcanthamoeba cysts.Rev InfDis 1991; 13: 418.

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