Determination of Amoebicidal Activities of Multipurpose Contact Lens ...

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Feb 4, 2003 - lens solutions against Acanthamoeba, Buck and colleagues (4) found that of ..... 24 h, let alone the MMRDT; however, after 24 h of exposure all solutions .... Hugo, E. R., W. R. McLaughlin, K. H. Oh, and O. H. Tuovinen. 1991.

JOURNAL OF CLINICAL MICROBIOLOGY, July 2003, p. 2992–3000 0095-1137/03/$08.00⫹0 DOI: 10.1128/JCM.41.7.2992–3000.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 41, No. 7

Determination of Amoebicidal Activities of Multipurpose Contact Lens Solutions by Using a Most Probable Number Enumeration Technique Tara K. Beattie,1* David V. Seal,2 Alan Tomlinson,1 Angus K. McFadyen,3 and Anthony M. Grimason4 Department of Vision Sciences1 and Department of Mathematics,3 Glasgow Caledonian University, and Environmental Health Division, Department of Civil Engineering, University of Strathclyde,4 Glasgow, Scotland, and Applied Vision Research Centre, City University, London, United Kingdom2 Received 4 February 2003/Returned for modification 23 March 2003/Accepted 6 April 2003

Six multipurpose contact lens solutions [All-in-One, All-in-One (Light), ReNu MultiPlus, Optifree Express, Complete, and Solo-care soft] were tested for their efficacies against Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique for amoebic enumeration. Against trophozoites, All-in-One, ReNu Multiplus, and Optifree Express achieved total kill (log reduction of >3) after the manufacturer’s minimum recommended disinfection time (MMRDT), with the remaining solutions failing to reach a log reduction of 1. After 24 h of exposure, all solutions proved trophozoiticidal, achieving, with the exception of Complete (log reduction of 3.13), total kill. Against cysts, All-in-One gave a log reduction of >3 within the MMRDT, with all other solutions failing to achieve a log reduction of 1. After 24 h of exposure, All-in-One achieved total kill of cysts (log reduction of 3.74), ReNu MultiPlus gave a log reduction of 3.15, and the remaining solutions reached log reductions of between 1.09 and 2.27. The MPN technique provides a simple, reliable, and reproducible method of amoebic enumeration that depends on simply establishing the presence or absence of growth on culture plates inoculated with a series of dilutions and determining the MPN of amoebae present from statistical tables. By use of this technique, two of the multipurpose solutions tested, ReNu MultiPlus and Optifree Express, demonstrated effective trophozoiticidal activities within the recommended disinfection times; however, only All-in-One proved effective against both trophozoites and cysts over the same time period. This MPN technique, which uses axenically produced trophozoites and mature, doublewalled cysts, has the potential to form the basis of a national standard for amoebicidal efficacy testing of multipurpose contact lens disinfecting solutions. being rinsed with tap water containing Acanthamoeba. The organisms in turn attach to the lens, which acts as a mechanical vector, transmitting the amoebae onto the corneal surface, where invasion and subsequent infection can occur. The use of ineffective lens disinfection systems (49), homemade saline (48), and tap water (44, 46) and contamination of lens storage cases (10, 15, 25) have all been cited as important risk factors for the disease. Initially, the incidence of AK among contact lens wearers (CLWs) was thought to be low. However, in 1996 Mathers and colleagues (29) used tandem scanning confocal microscopy to screen the infected eyes of 217 patients with keratitis for the presence of Acanthamoeba. The organism was suspected in 51 patients, and the presence of the organism was confirmed by cytology in 43 of them. This led the investigators to conclude that the marked increase in acanthamoebic detection by tandem scanning confocal microscopy strongly suggested that the disease was more prevalent than initially suspected. A cohort study in Scotland quoted a peak incidence of AK in 1995 of 1 in 6,720 CLWs (45). Similar studies in Holland in 1996 (5) and Hong Kong during 1997 and 1998 (24) gave annual AK incidence rates of 1 in 200,000 CLWs and 1 in 33,000 CLWs, respectively. The popularity of rigid gas-permeable contact lenses in Holland is the main reason for the low incidence found in that country. Radford and colleagues (39, 40) carried out three multicenter questionnaire surveys on AK,

Acanthamoeba is a genus of free-living protozoa with a widespread distribution in the environment. Organisms of this genus are commonly found inhabiting soil (8) and aquatic environments (9, 23), but they have also been isolated from swimming pools (32), tap water (44, 46), bottled mineral water (37), atmospheric samples (22), and even contact lens care solutions (47). The organisms’ life cycle is composed of two distinct stages: a motile, metabolically active trophozoite stage in which the organism is capable of multiplication and is sensitive to noxious stimuli, and a dormant cyst stage, in which the organism is resistant to desiccation, disinfection, and extremes of temperature. Ocular infections due to Acanthamoeba were first reported in the early 1970s (18, 34), but it was not until the mid-1980s that a connection between contact lens wear and disease was established (33). Ledee and colleagues (27) demonstrated a direct chain of causation of Acanthamoeba keratitis (AK) using DNA matching of isolates of Acanthamoeba griffini from the corneal scraping of an infected individual, the individual’s lens storage case, and the individual’s bathroom water supply. In such an incident, the storage case becomes contaminated after

* Corresponding author. Mailing address: Department of Vision Sciences, City Campus, Glasgow Caledonian University, Cowcaddens Rd., Glasgow G4, Scotland. Phone: 44 141 331 3695. Fax: 44 141 331 3387. E-mail: [email protected] 2992

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first in England from 1992 to 1996 and then in England and Wales during 1997–1998 and 1998–1999, giving incidence rates of 1 case in 39,370 CLWs (39), 1 case in 47,620 CLWs (40), and 1 case in 55,555 CLWs (40), respectively. The data from the second study have recently been corrected to 1 in 32,260 and 1 in 37,040, respectively, by Seal et al. (D. V. Seal, A. Tomlinson, T. K. Beattie, D. Fan, and E. Wong, Letter, Br. J. Ophthalmol. 87:516–517, 2003) due to underreporting in the original survey (40). It is thought that the introduction of multipurpose cleaning and disinfecting solutions has been a major factor contributing to the reduction in the frequency of this disease (49). The increased recognition of contact lens-associated AK during the late 1980s and early 1990s resulted in several investigations into the amoebicidal effects of contact lens solutions. However, a major hurdle to be overcome when carrying out such a study is the enumeration of the amoeba. In a recent review of methods used to evaluate the effectiveness of contact lens solutions against Acanthamoeba, Buck and colleagues (4) found that of the studies reviewed, 30% used no quantitative method and merely reported the presence or absence of viable amoebae. Several quantitative methods have been used, such as direct counting with a hemocytometer (2, 6), standard plaque assay (16, 19), a quantitative microtiter method (2), and enumeration of track-forming units developed on nonnutrient agar with a bacterial overlay (20). With the exception of one study (38), a technique of organism enumeration that has been largely overlooked is the most probable number (MPN) technique, which is a means for estimating, without any direct counting, the density of organisms present in a liquid. The MPN technique was first introduced by McCrady (30, 31) and consists of the incubation of aliquots of sample dilutions with appropriate media at an ideal temperature for the desired organism. After incubation, the aliquots are examined for the presence or absence of growth (with growth indicating the presence of at least one organism in that aliquot). Initially, mathematical equations for estimating the number of organisms present, based on the number of aliquots showing growth, were solved approximately (30, 31). Computers have now been used to develop more accurate MPN tables (51, 52). Previous methods of Acanthamoeba enumeration, such as direct counting and plaque assays, can be time-consuming and labor-intensive, and some methods may not distinguish between viable and nonviable amoebae. The present study used a simple enumeration technique, the MPN technique, to determine the efficacies of six multipurpose contact lens solutions against Acanthamoeba castellanii trophozoites and cysts. MATERIALS AND METHODS Organism. A. castellanii was chosen as the test organism, as it is the best indicator Acanthamoeba species of molecular type T4, typical of strains causing AK (T. K. Beattie, A. Tomlinson, D. V. Seal, Letter, Br. J. Ophthalmol. 86: 1319–1320, 2002). An axenic culture of A. castellanii 1501/1A was obtained from the Culture Collection of Algae and Protozoa (Freshwater Biological Association, The Ferry House, Ambleside, United Kingdom) and maintained axenically in tissue culture flasks containing proteose peptone glucose broth (PPG) (36). Trophozoites for experimental use were produced by subculturing amoebae in 75-cm2 tissue culture flasks containing 30 ml of PPG and incubating at 32°C for 2 days. After incubation the PPG was replaced with Page’s amoebic saline (PAS) (36), and a sterile cell scraper was used to gently remove the trophozoites that adhered to the base of the tissue culture flask. The PAS containing the trophozoites was then centrifuged at 3,000 rpm for 10 min at room temperature, the


supernatant was removed, and the Acanthamoeba pellet was resuspended in PAS. To produce mature cysts, the amoebae were subcultured into fresh 75-cm2 tissue culture flasks containing 30 ml of PPG and incubated at 32°C for 4 days. After incubation, the trophozoites were removed as described above and transferred to fresh nonnutrient agar (NNA) plates (36) without bacteria. The plates were sealed with Parafilm and incubated for 10 to 14 days at 32°C. After incubation the cysts were washed off the NNA plates with PAS. Saline cultures of trophozoites or cysts were enumerated with a Neubauer hemocytometer and adjusted to 106 trophozoites or cysts/ml by dilution or centrifugation. Procedure. Trophozoites or cysts were exposed to six multipurpose solutions (MPSs) and a control solution (0.9% PAS). The MPSs used were All-in-One, All in One (Light), ReNu Multiplus, Optifree Express, Solo-care soft, and Complete (see Table 1 for the manufacturers and ingredients). Aliquots (0.1 ml) of trophozoite or cyst culture were added to 9.9 ml of each MPS and the control in sterile glass universal bottles to give initial concentrations of 104 trophozoites or cysts/ml. The universal bottles were vortexed to distribute the organisms homogeneously throughout the MPSs or the control solution. All universal bottles were placed on an orbital shaker and shaken at 80 rpm in a 25°C incubator to maintain a constant temperature throughout the disinfecting period. At predefined intervals (0, 2, 4, 8, and 24 h), 1-ml aliquots were removed from each MPS and the control solution and placed in 9 ml of Dey Engley (DE) broth (Difco) for disinfectant neutralization. From this 1 in 10 dilution, a 1 in 100 dilution was prepared in PAS, and from this dilution, five aliquots each of 1, 0.1, and 0.01 ml were inoculated onto NNA plates seeded with heat-killed Klebsiella aerogenes (WPRL CN345). The wells of flat-bottom 6-well tissue culture plates were used for the 1-ml aliquots, and the wells of 12-well plates were used for the 0.1- and 0.01-ml aliquots. All plates were sealed and incubated at 32°C. The plates were examined for the presence of viable trophozoites after 3 and 7 days of incubation. A plate showing amoebic growth was scored as 1; no growth was scored as 0. The score for each of the 10-fold dilutions (i.e., 1, 0.1, and 0.01 ml) gave one value of a three-digit number, which was entered into the MPN table (Table 2) to give the most probable number of amoebae per milliliter of MPS or the control solution at each time interval. For example, five positive plates with 1 ml, four positive plates with 0.1 ml, and two positive plates with 0.01 ml give a three-digit number of 542, which, when entered into Table 2, gives an MPN of 2,200 amoebae/ml. Five 1-ml aliquots of the 1 in 10 dilution prepared in DE broth were also inoculated onto 90-mm petri dishes containing NNA seeded with heat-killed bacteria; petri dishes were used, as the colored DE broth obscured the amoebae in the six-well tissue culture plates. If no amoebic growth was detected in the aliquots taken from the 1 in 100 dilution, then the 1 in 10 DE broth dilution plates were checked for growth; no growth indicated total kill of the trophozoites and cysts. Each MPS was tested in triplicate against trophozoites and cysts. By using the MPN of trophozoites or cysts per milliliter of MPS, the average log reductions over the manufacturer’s minimum recommended disinfection time (MMRDT) and 24 h were determined. A one-factor, balanced analysis of variance was used to analyze the data, with Tukey’s pairwise comparison used for follow-up testing. The effect of exposure to DE disinfectant neutralization broth on the trophozoites and cysts was assessed by adding 0.1-ml aliquots of a trophozoite or cyst culture to 9.9 ml of DE broth in sterile glass universal bottles, again giving an initial concentration of 104 trophozoites or cysts/ml. The DE broth containing the trophozoites or cysts was then processed as described above for the MPSs and the control solution. It has been reported in the literature (53) that some of the active ingredients found in the MPSs under investigation, namely, Aldox (myristamido propyldimethylamine) and polyhexamethylene biguanide (PHMB), can adhere to certain types of plastics and glass, respectively. A preliminary test was therefore carried out with two of the test solutions, Optifree Express (which contains Aldox) and All-in-One (which contains PHMB), to determine if the testing vessel had any effect on the efficacies of the solutions. Aliquots (0.1 ml) of the cyst culture were added to 9.9 ml of Optifree Express in either a glass bottle, a plastic bottle, or a plastic bottle that had been soaked overnight in Optifree Express or to 9.9 ml of ReNu Multiplus in a glass bottle, a plastic bottle, or a glass bottle that had been soaked overnight in ReNu Multiplus. The MPSs in the various bottles were processed as described above for all MPSs and the control solution.

RESULTS The average and standard deviation log10 MPN at 0 h, after the MMRDT, and after 24 h for the three trophozoite and cyst

EDTA (0.02%)


Allergan Complete


Ciba Vision Solo-care soft

PHMB and polyhexanide are the same preservative. EDTA and disodium edetate are the same chelating agent.

Poloxamer (0.05%), hydroxypropyl methylcellulose (0.15%)

Alcon Optifree Express

0.0001% PHMB

Poloxamer 407

Disodium hydrogen phosphate Sodium phosphate

Disodium edetate (0.025%)

Poloxamine citrate Borate, sorbitol aminomethyl propanol

0.001% polyquad (polidronium chloride), 0.0005% Aldox (myristamidopropyl dimethylamine) 0.0001% polyhexanide

Sauflon Sauflon Bausch & Lomb All-in-One All-in-One (Light) Renu MultiPlus

Poloxamine Poloxamer Poloxamine (1%) Phosphate Phosphate Sodium borate, boric acid 0.0005% polyhexanide 0.0001% polyhexanide 0.0001% polyhexanide

Cleaning or lubricating agent Buffer system Manufacturer

TABLE 1. MPS ingredients and manufacturers




Disodium edetate (0.1%) Disodium edetate (0.1%) Hydranate (hydroxyalkyl phosphonate) (0.03%), EDTA (0.1%) EDTA (0.5%)


Chelating agentb


replicates in all test MPSs and the control are shown in Table 3. No significant difference was detected between any of the MPS trophozoite and cyst replicates from each of the test runs, demonstrating the reliability of the MPN method for enumeration. A significant difference was detected between two of the trophozoite control replicates (P ⫽ 0.032) and two of the cyst control replicates (P ⬍ 0.001); this was due to low inocula in two of the replicates; however, the amoebic numbers within each control replicate remained constant. The average log reductions for trophozoites and cysts challenged with the six MPSs over a 24-h period are presented in Fig. 1 and 2, respectively. Table 3 gives the average log reduction in the viabilities of the trophozoites and cysts after the MMRDT and 24 h. The effects of three MPSs (All-in-One, ReNu Multiplus, and Optifree Express) were found to differ significantly (P ⬍ 0.001) from those of the control solution and the other three MPSs after their MMRDTs (4 h for All-in-One and ReNu, 6 h for Optifree), achieving total kill of trophozoites (log reduction ⬎3). The remaining MPSs [All-in-One (Light), Complete, Solo-care soft] failed to have any impact on the trophozoites by their MMRDTs [4 h for All-in-One (Light) and Complete, 10 min for Solo-care soft], with no significant difference from the effect of the control solution being detected. However, by 24 h all solutions showed significant (P ⬍ 0.001) trophozoiticidal effects. All-in-One demonstrated cysticidal activity within the MMRDT, giving a log reduction in viability of 3.44, with this being significantly different from the reductions for the control solution and the other MPSs (P ⬍ 0.001). With the exception of ReNu Multiplus, the remaining solutions had no cysticidal effect by their MMRDTs; ReNu Multiplus gave a log reduction of 0.98, but this result was not statistically significant. By 24 h, only All-in-One and ReNu Multiplus demonstrated effects significantly different from that of the control solution (P ⬍ 0.001); the remaining solutions achieved log reductions in cyst viability of between 1.09 and 2.27, but these were not found to be statistically significant. Exposure to DE disinfectant neutralization broth for 24 h was found to have no detrimental effect on the amoebae. By using a three-factor analysis of variance, no significant difference in amoebic numbers was detected when two of the solutions, ReNu Multiplus and Optifree Express, were tested in glass, plastic, or MPS-soaked glass and plastic bottles (Fig. 3). DISCUSSION The International Organization for Standardization (ISO) has produced standards by which contact lens disinfecting solutions are assessed. To meet the primary criteria of “microbiological requirements and test methods for products and regimens for hygienic management of contact lenses” (17), solutions must achieve a 3-log reduction with each of three species of bacteria and a 1-log reduction with each of two species of fungi by the MMRDT. The standard does not require solutions to be tested against Acanthamoeba; therefore, the solutions tested in the present study have no requirement to be acanthamoebicidal. The reasons that ISO gives for exclusion of Acanthamoeba in its standard are that no standard method to test the efficacies of solutions against Acanthamoeba

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TABLE 2. Table for estimation of MPNs of Acanthamoeba per milliliter of test multipurpose solutiona No. of positive aliquots from DE disinfectant neutralization brothc 1 ml

0.1 ml

0.01 ml

0 0 0 1 1 1 1 2 2 2 2 2 2 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4

0 0 1 0 0 1 2 0 0 1 1 2 3 0 0 1 1 2 2 3 0 0 1 1 2 2 3 3

0 1 0 0 1 0 0 0 1 0 1 0 0 0 1 0 1 0 1 0 0 1 0 1 0 1 0 1

a b c


⬍1 20 20 20 40 40 50 40 50 50 70 70 110 70 90 90 130 130 160 160 110 140 160 200 200 250 250 310

Most probable range (95% CI)b

⬍1 20 20 20 40 40 50 40 50 50 70 70–90 110 70 90 90 130 130 140–160 140–160 110–130 140–160 140–160 180–200 180–220 230–270 230–270 290–340

No. of positive aliquots from DE disinfectant neutralization brothc 1 ml

0.1 ml

0.01 ml

4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

4 4 0 0 0 1 1 1 1 2 2 2 2 3 3 3 3 3 4 4 4 4 4 5 5 5 5 5 5

0 1 0 1 2 0 1 2 3 0 1 2 3 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 5


Most probable range (95% CI)

320 380 220 290 410 310 430 600 850 500 700 950 1,200 750 1,100 1,400 1,750 2,100 1,300 1,700 2,200 2,800 3,450 2,400 3,500 5,400 9,100 16,000 ⬎18,000

290–340 340–410 200–230 250–340 360–500 270–360 360–500 500–700 700–950 400–550 600–800 800–1,100 1,050–1,350 650–900 900–1,250 1,200–1,600 1,550–2,000 1,850–2,400 1,100–1,500 1,500–2,000 1,900–2,500 2,400–3,200 3,000–3,900 2,000–2,800 2,900–4,200 4,500–6,600 7,500–11,000 13,500–19,000 ⬎18,000

The table was adopted from the work of Tillett (52). The most probable range gives counts that are at least 95% as likely as the MPN. CI, confidence interval. Five aliquots of each dilution were tested.

is available, no standard method of amoeba recovery or enumeration is available, and no species type has specifically been identified. The ISO considers the reduction of bacteria present within the lens case with an appropriate bactericidal care solution to be an adequate measure for prevention of Acanthamoeba contamination and multiplication within the case by removal of the food source for the amoebae. However, as no minimum infective dose for acanthamoebic infection has been determined, the presence of any Acanthamoeba within a contact lens case may place the wearer at risk of infection. Of the MPSs tested, only All-in-One achieved a 3-log reduc-

tion for both trophozoites and cysts within the MMRDT, with this reduction being the level of killing that the ISO standard would expect a solution to provide against bacteria. Both ReNu Multiplus and Optifree Express achieved 3-log reductions against trophozoites by their MMRDTs. As cysts are more resistant to disinfection than trophozoites (12, 14, 26), this result is of less relevance, as neither solution was effectively cysticidal over the same time scale. The remaining solutions were neither trophozoiticidal nor cysticidal within their MMRDTs. However, after 24 h all solutions killed at least 99.9% of the trophozoites. ReNu Multiplus proved to be cys-

TABLE 3. Average and standard deviation log10 counts of Acanthamoeba trophozoite and cyst replicatesa at the start of the experiment (0 h), after the MMRDTs (see Results for times), and after 24 h and average log reductions for trophozoites and cysts over the MMRDT and 24 h MPS

Control All-in-One All-in-One (Light) ReNu Multiplus Optifree Express Complete Solo-care soft a b c

Avg (SD) log10 count at 0 h

Avg (SD) log10 count after MMRDT




3.67 (0.11) 3.10 (0.36) 3.63 (0.30) 3.49 (0.09) 3.82 (0.34) 3.89 (0.27) 3.98 (0.38)

3.60 (0.44) 3.74 (0.21) 3.58 (0.48) 3.68 (0.24) 3.60 (0.11) 3.74 (0.21) 3.74 (0.21)

3.63 (0.30) 0 (0) 3.46 (0.97) 0 (0) 0 (0) 3.27 (0.47) 3.91 (0.37)

Three replicates each of trophozoites and cysts were tested. Trophs, trophozoites. TK, total kill.


3.54 (0.20) 0.30 (0) 3.46 (0.32) 2.70 (0.96) 3.42 (0.49) 3.73 (0) 3.73 (0.19)

Log reduction over MMRDT

Avg (SD) log10 count after 24 h

Log reduction over 24 h







0.04 3.10 (TK:c) 0.17 3.49 (TK) 3.82 (TK) 0.62 0.07

0.06 3.44 0.12 0.98 0.18 0.01 0.01

3.90 0 (0) 0 (0) 0 (0) 0 (0) 0.76 (0.69) 0 (0)

3.68 0 (0) 2.00 (0.97) 0.53 (0.92) 2.37 (0.68) 2.65 (0.41) 1.47 (1.56)

⫺0.23 3.10 (TK) 3.63 (TK) 3.49 (TK) 3.82 (TK) 3.13 3.98 (TK)

⫺0.08 3.74 (TK) 1.58 3.15 1.23 1.09 2.27




FIG. 1. Average trophozoiticidal effects of MPSs over 24 h (n ⫽ 3).

ticidal after 24 h, killing 99.9% of the cysts, with the remaining solutions achieving log reductions of between 1.09 and 2.27. The MPN method has been used extensively for the microbiological examination of water, but more recently, it has been adapted to suit the enumeration of a variety of organisms other than bacteria, for example, the enumeration of viable planktonic diatoms from sediment samples (13) and a variety of protozoa from soils and infiltration systems (11, 50). In 1995, Perrine et al. (38) used the MPN technique to enumerate Acanthamoeba in studies determining the amoebicidal efficien-

cies of various diamidines against two strains of Acanthamoeba polyphaga; MPNs were read off tables published by the American Public Health Association (1) without modification. However, the investigators gave no detailed comment on the actual usefulness of this method when it was applied to the enumeration of Acanthamoeba. In the present study the MPN technique was found to be a simple method which, when used repeatedly, gave consistent determinations of the amoebicidal activities of various MPSs. Although the present study investigated only MPSs, work is planned to use this method to

FIG. 2. Average cysticidal effects of MPSs over 24 h (n ⫽ 3).

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FIG. 3. Cysticidal effects of ReNu Multiplus and Optifree Express in plastic, glass, and MPS-soaked bottles over 24 h.

determine the amoebicidal activities of one- and two-step hydrogen peroxide systems. Buck et al. (4) concluded their review of the methods used to evaluate the effectiveness of contact lens solutions against Acanthamoeba by listing key procedures that should be included in any “standard” method used to investigate the efficacy of a contact lens solution. First, a species of Acanthamoeba associated with keratitis, i.e., A. castellanii or A. polyphaga, should be used. Also, a strain that grows well axenically and that produces enough organisms to allow detection of antiamoebic activity and comparison of the activity with that of a control solution should be used. An appropriate neutralization step should be included to cease the activity of the contact lens solution; the toxicity of the neutralizer should also be assessed. Finally, an appropriate recovery method that determines viability and that quantifies the surviving organisms should be used. The present MPN protocol fulfills all of the criteria mentioned above. A. castellanii, one of the most common causative agents of AK, was selected as the test organism. The amoebae were grown axenically and produced abundant trophozoites, and encystation on NNA plates in the absence of bacteria produced abundant, mature, double-walled cysts. MPS disinfection was brought to an end point with DE neutralization broth, which, when tested, as reported by Buck and Rosenthal (2), was found to have no toxic effect on the amoebae. Finally, the MPN technique provided a simple method that determined viability and enumerated the surviving organisms by merely establishing the presence or absence of amoebic growth on culture plates derived from three 10-fold dilutions cultured five times and reading the numbers present from preexisting, albeit adapted, MPN tables. We believe that this new method incorporating MPN enumeration provides the basis for a standardized amoebicidal activity test for contact lens solutions. With the exception of Optifree Express, all solutions contain the preservative PHMB, with a concentration of 5 ppm in

All-in-One and a concentration of 1 ppm in each of the remaining solutions. Several studies have investigated both the minimum trophozoite amoebicidal concentration (MTAC) and the minimum cysticidal concentration (MCC) of PHMB. Larkin et al. (26) tested the in vitro sensitivities of corneal isolates of Acanthamoeba to various drugs and found the MTAC of PHMB to be 0.87 ␮g/ml (range, 0.49 to 1.49 ␮g/ml for five isolates) and the MCC to be 2.11 ␮g/ml (range, 0.97 to 3.9 ␮g/ml for five isolates) after 48 h of exposure. In a similar study, also with corneal isolates of Acanthamoeba, Hay et al. (14) demonstrated the MTAC of PHMB to be 1 ␮g/ml and the MCC to be 3 ␮g/ml after 48 h of exposure. A third study carried out by Elder et al. (12) found the MTAC of PHMB to be 1.3 ␮g/ml and the MCC to be 2.2 ␮g/ml after 48 h of exposure. It was therefore not unexpected that All-in-One, which contains 5 ␮g of PHMB per ml (5 ppm), was both trophozoiticidal and cysticidal after 24 h of exposure; however, the solution also effectively killed both trophozoites and cysts within the MMRDT of 4 h. The remaining solutions containing 1 ␮g of PHMB per ml (1 ppm) would have been expected to demonstrate some trophozoiticidal effect after 24 h of exposure, but may not have been expected to produce total kill of trophozoites, which all solutions except Complete (3.13-log reduction) achieved. The solutions containing 1 ppm PHMB would not have been expected to produce a cysticidal effect by 24 h, let alone the MMRDT; however, after 24 h of exposure all solutions except ReNu Multiplus produced log reductions in viable amoebae of between 1.09 and 2.27; ReNu Multiplus killed ⬎99.9% of the cysts. Alcon, the manufacturer of Optifree Express (which contains Polyquad and Aldox), has carried out several studies into the amoebicidal activity of their MPS (3, 42, 43). After 6 h of exposure, Optifree Express was found to produce log reductions in viability of 1.3 to 4.8 for trophozoites and 2 to 3.2 for cysts of various species and strains of Acanthamoeba (3, 42, 43). In an independent study carried out by Kilvington (20),



again, with various species of Acanthamoeba, including A. castellanii and A. polyphaga, Optifree Express achieved log reductions in viability of approximately 4 for trophozoites and 2 to 3 for cysts after the MMRDT. However, Kilvington and Anger (21) recently investigated the efficacy of Optifree Express against cysts of A. polyphaga produced by different methods, i.e., the defined, constant-pH encystment medium of Neff et al. (35) or NNA plates seeded with Escherichia coli, and also at different stages of maturity. They found that regardless of the method used to produce mature cysts (7 days of incubation for both methods), exposure to Optifree Express had little effect, achieving log reductions of only 0.3 to 0.5. Log reductions in viability of 1.1 to 2 were, however, achieved with immature cysts produced after 0.5 and 1 day of incubation in the defined, constant-pH encystment medium of Neff et al. (35). MowreyMcKee (M. F. Mowery-McKee, Abstr. Assoc. Res. Vision Ophthalmol., abstr. 3084, 2002) also demonstrated that Optifree Express had a limited amoebicidal effect after the recommended disinfection time of 6 h when it was tested against a strain of A. castellanii, giving log reductions of 2.5 and 0.5 for trophozoites and cysts, respectively. In the present study, after 6 h of exposure, Optifree Express produced average log reductions of 3.82 and 0.18 for trophozoites and cysts, respectively. During their review of the methods used to evaluate the effectiveness of contact lens care solutions, Buck et al. (4) suggested that the seemingly contradictory results in the activities of contact lens solutions against Acanthamoeba were due in part to variations in the methodologies used. They included organism strain, cyst production, inoculum preparation, neutralization of the test solution, recovery, quantification method, and determination of the viability of survivors as variables that may be responsible for the dissimilarities in the results between studies (4). Kilvington and Anger (21) suggested that the difference in results found between their study conducted in 2001 and the previous work (20) may have been due to the different methods of cyst production. However, they reported in their 2001 study (21) that the method of cyst production used in that study had no effect on the efficacies of the lens solutions tested; only the stage of cyst maturity altered the efficacies. In the present study, incubation of trophozoites in the defined, constant-pH encystment medium of Neff et al. (35) produced many rounded trophozoites and immature cysts, but failed to produce double-walled mature cysts, even after lengthy periods of incubation (4 or more weeks). To overcome this problem, we reverted to producing cysts using 10 to 14 days of incubation on NNA plates. The use of axenically grown organisms in efficacy studies omits the problem of unwanted organic matter, i.e., dead or live bacteria, in the initial inoculum. This benefit would be lost if bacteria were then introduced during the encystment stage, and so no bacteria were added to the NNA plates. Cysts were therefore produced by axenically growing the trophozoites in PPG for 4 days before the organisms were transferred to NNA plates for 10 to 14 days of incubation. Examination of the cysts after this time revealed mature cysts with clearly visible endocyst and exocyst walls. Van Duzee and Schlech (53) reported that the activities of various ingredients of contact lens solutions can be affected by the container in which testing is carried out. One of the active ingredients in Optifree Express, Aldox, adheres to certain


types of plastics, and PHMB, the active ingredient in the remaining solutions, adheres to glass. Therefore, prior to commencement of the main study, two of the MPSs under investigation, Optifree Express (which contains Aldox) and All-inOne (which contains PHMB), were tested in both plastic and glass bottles. Optifree Express was also tested in a plastic bottle that had been soaked overnight in Optifree Express, and ReNu Multiplus was tested in a glass bottle soaked overnight in ReNu Multiplus. No significant differences in amoebic numbers were detected in the various testing bottles with the two solutions. As neither plastic nor glass was found to affect efficacy testing, the remainder of the study was conducted with glass universal bottles. In order for a disinfectant like PHMB to be cysticidal, it must gain access to the trophozoite internalized within the cyst. The most obvious route is via the ostioles or the pores in the double cell wall that connect the outer exocyst and inner endocyst, thus allowing the internalized amoeba to communicate with its outside environment. It is believed that the ostioles are plugged with mucopolysaccharide, which would have to be destabilized to allow penetration of the disinfectant. The effectiveness of the higher concentration of PHMB (5 ppm), found in All-in-One, is thought to be due to the binding of this highly positively charged molecule to the mucopolysaccharide, resulting in penetration and irreversible damage to the cell membrane and the cell contents. The cell damage caused by PHMB is associated first of all with leakage of calcium ions from the plasma membrane. This factor may be potentiated by the chelating agent hydranate in ReNu Multiplus, which would explain the enhanced trophozoiticidal and cysticidal actions of this MPS compared to those of the other solutions containing 1 ppm PHMB. Rapid chelation of the calcium ions outside the cell hastens further leakage from within the cell, leading to protein disruption and, ultimately, irreversible cellular damage. An alternative hypothesis is the inclusion of sodium borate and boric acid in the formulation of ReNu Multiplus, which has been shown to potentiate the activity of PHMB by twofold, although EDTA at 0.1% (wt/vol), which is also present in ReNu Multiplus, has been found to inhibit activity fourfold (19). Since the PHMB concentration used (1 ppm [1 ␮g/ml]) is at its minimum effective level (MTAC), the effects of the formulation could be crucial and could explain the differences in activities of the various commercial MPSs with the same concentration of PHMB (1 ppm) that were found. It has been established that a contact lens can act as a mechanical vector for transportation of Acanthamoeba present in the storage case onto the corneal surface, where subsequent multiplication and invasion of the corneal tissue may occur, ultimately leading to keratitis (27). It is not yet known if Acanthamoeba transported to the eye on a contact lens puts the wearer at increased risk of other acanthamoebic infections. Acanthamoeba transferred to the eye via a contact lens will most likely reach the nasal cavity via lacrimal drainage. In a healthy individual this should not cause a problem, as Acanthamoeba can exist as a commensal organism in the nasopharynges of apparently healthy individuals (41). However, it is yet to be established if the presence of Acanthamoeba within the nasopharynx of an immunocompromised individual increases the risk of granulomatous amoebic encephalitis, a usually fatal disease of the central nervous system (CNS) thought to be

VOL. 41, 2003


caused by the hematogenous spread of Acanthamoeba from a primary focus in the lower respiratory tract, skin, or open wounds (28). Culbertson and colleagues (7) proved, using mice, that Acanthamoeba can reach the CNS directly via the olfactory neuroepithelium within the nasal cavity in a fashion similar to that of Naegleria fowleri, the etiological agent of fulminant primary amoebic meningoencephalitis, an infection of the CNS similar to that caused by Acanthamoeba. Jones et al. (18) reported a case of Acanthamoeba uveitis associated with fatal meningoencephalitis. With an ever increasing patient population immunocompromised due to human immunodeficiency virus infection and AIDS, transplantation, cancer, and corticosteroid treatment, it would seem prudent to produce contact lens care solutions that protect the lens wearer, whether they be immunocompromised or not, from any risk of infection by Acanthamoeba. The MPN-based efficacy test for MPSs conducted in the present study using axenically cultured A. castellanii trophozoites and mature cysts produced on NNA plates in the absence of bacteria proves to be a simple, reliable, and reproducible technique that we consider suitable for the basis of a standardized efficacy test to which multipurpose contact lens solutions should be subjected. CLWs should be confident that the MPSs that they have been recommended to use protect them from possible serious infections with this organism. ACKNOWLEDGMENT We are most grateful to Sauflon Pharmaceuticals, which partially funded the development of the MPN method. REFERENCES 1. American Public Health Association. 1971. Standard methods for the examination of water and wastewater, 13th ed., p. 657. American Public Health Association, Washington, D.C. 2. Buck, S. L., and R. A. Rosenthal. 1996. A quantitative method to evaluate neutralizer toxicity against Acanthamoeba castellanii. Appl. Environ. Microbiol. 62:3521–3526. 3. Buck, S. L., R. A. Rosenthal, and R. L. Abshire. 1998. Amoebicidal activity of a preserved contact lens multipurpose disinfecting solution compared to a disinfection/neutralisation peroxide system. Contact Lens Anterior Eye 21: 81–84. 4. Buck, S. L., R. A. Rosenthal, and B. A. Schlech. 2000. Methods used to evaluate the effectiveness of contact lens care solutions and other compounds against Acanthamoeba: a review of the literature. CLAO J. 26:72–84. 5. Cheng, K. H., S. L. Leung, S. L., H. W. Hoekman, W. H. Beekhuis, P. J. H. Mulder, and A. J. M. Geerards. 1999. Incidence of contact-lens-associated microbial keratitis and its related morbidity. Lancet 354:181–185. 6. Connor, C. G., S. L. Hopkins, and R. D. Salisbury. 1991. Effectivity of contact lens disinfection systems against Acanthamoeba culbertsoni. Optom. Vis. Sci. 68:138–141. 7. Culbertson, C. G., J. W. Smith, H. K. Cohen, and J. R. Minner. 1959. Experimental infection of mice and monkeys by Acanthamoeba. Am. J. Pathol. 35:185–197. 8. Culbertson, C. G. 1971. The pathogenicity of soil amoebas. Annu. Rev. Microbiol. 25:231–254. 9. Davies, P. G., D. A. Caron, and J. M. Sieburth. 1978. Oceanic amoebas from the North Atlantic: culture, distribution and taxonomy. Trans. Am. Microsc. Soc. 97:73–88. 10. Devonshire, P., F. A. Munro, C. Abernethy, and B. J. Clark. 1993. Microbial contamination of contact lens cases in the west of Scotland. Br. J. Ophthalmol. 77:41–45. 11. Ekelund, F. 1998. Enumeration and abundance of mycophagous protozoa in soil, with special emphasis on heterotrophic flagellates. Soil Biol. Biochem. 30:1343–1347. 12. Elder, M. J., S. Kilvington, and J. K. G. Dart. 1994. A clinicopathologic study of in vitro sensitivity testing and Acanthamoeba keratitis. Investig. Ophthalmol. Vis. Sci. 35:1059–1064. 13. Harris, A. S. D., K. J. Jones, and J. Lewis. 1998. An assessment of the accuracy and reproducibility of the most probable number (MPN) technique in estimating numbers of nutrient stresses diatoms in sediment samples. J. Exp. Mar. Biol. Ecol. 231:21–30.


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