MATERIALS AND METHODS

Contact lens solutions

All tested contact lens storage solutions and their respective contents are listed in Table 1. All tested solutions were taken from their original wrappings and were used before their stated expiry date. According to the manufacturer's instructions for Titmus H₂O₂ the neutralisation of the hydrogen peroxide is achieved by soaking the contact lenses in a second solution (catalase 170 IU/ml) for 10 minutes before wearing. When applying Oxysept Comfort a catalytic tablet is put into the storage case together with the 3% hydrogen peroxide solution. A minimum contact lens soaking time of 6 hours is required.

Amoebae

Three different strains of Acanthamoeba spp, including two strains of morphological group II (A hatchetti 11DS, A castellanii 4CL)^15,16 and one strain of morphological group III isolated from the brain of an experimentally infected mouse (A lenticulata strain 72/2)¹⁷ were used in this study. Tests were performed on both cysts and trophozoites of each strain.

The amoebae were cultured in the absence of any other organisms in 150 cm² tissue culture flasks in 36 ml of PYG (proteose peptone-yeast extract-glucose). Trophozoites and cysts were harvested from liquid cultures by centrifugation (500 g for 7 minutes) and the pellet resuspended in sterile 0.9% NaCl. The amoebae were counted in a Bürker-Türk haemocytometer. Trophozoites and cysts of each strain at a concentration of 10³, 10⁴, and 10⁵ cells/ml were used in the tests.

Performance of the tests

Tests were performed in 24 well microtitre plates. One ml contact lens solution per well was applied. Oxysept Comfort tests were performed in 15 ml Falcon tubes using 6 ml solution. According to the manufacturer's instructions the neutralising tablet (catalase 1 mg per tablet) was added at the same time as the 3% hydrogen peroxide solution. Further, three dilutions (1:1, 1:2, 1:4) from each of the solutions were tested for their amoebicidal effects.

After a soaking time of 30 minutes and 8 hours (overnight soaking of contact lenses), respectively, 100 μl samples were inoculated onto non-nutrient agar plates covered with a lawn of Escherichia coli. The plate cultures were sealed and incubated at 30°C for 14 days. All experiments were carried out in triplicate. The control groups were performed with sterile 0.9% NaCl. Amoebic growth was observed daily by phase contrast microscopy.

RESULTS

The effect of the three contact lens storage solutions on the different Acanthamoeba strains subject to the concentration of cysts, soaking time, and dilution is shown in Tables 2 and 3. As expected, the sensitivity of Acanthamoeba trophozoites to the applied solutions was higher than that of the cysts. After a soaking time of 8 hours trophozoites of all strains were killed by all tested disinfectant solutions.

After a soaking time of 8 hours the two step hydrogen peroxide system Titmus H₂O₂ 0.6% showed the best amoebicidal effects. The cysts of the morphological group II (A hatchetti, A castellanii) were totally destroyed. Only the cysts of the morphological group III, A lenticulata, at a concentration of 10⁵ showed viability. Even in a dilution of 1:1 all cysts of morphological group II in all concentrations were completely destroyed. Cysts of A castellanii and A hatchetti were destroyed after exposure to this solution for 30 minutes (Table 2)

The multipurpose disinfecting solution Meni Care Plus also has amoebicide effects. However, cysts of the strain A lenticulata (morphological group III) and A hatchetti (morphological group II) at a concentration of 10⁵ could not be completely destroyed.

One step Oxysept Comfort was less effective against Acanthamoeba cysts. After a soaking time of 8 hours cysts of all tested strains were still viable.

A lenticulata (72/2) belonging to morphological group III was found to be the most resistant strain in comparison with the other isolates tested.

DISCUSSION

We have shown that the two step hydrogen peroxide system 0.6% Titmus H₂O₂, after an exposure time of 8 hours (overnight soaking of contact lenses), is very effective against Acanthamoeba.

The disinfecting ability of hydrogen peroxide is directly proportional to its length of exposure to the micro-organism: an incubation of 2–3 hours of exposure time to 3% H₂O₂ is sufficient to kill bacteria, HIV, fungi, and Acanthamoeba.^18,19 However 3% H₂O₂ Oxysept Comfort with a neutralisation tablet (1 mg catalase per tablet) was not effective against cysts of all three species in the lowest concentration even after 8 hours of soaking time. Because of the toxic effects of hydrogen peroxide to the conjunctival and corneal epithelium it must be neutralised before contact lens wear. Therefore, one step systems often use a catalyst (platinum ring, tablet) to neutralise the H₂O₂, producing oxygen and water. Presumably, the catalytic tablet of the Oxysept Comfort neutralises the 3% hydrogen peroxide too quickly to guarantee sufficient soaking time and effective killing of the Acanthamoeba cysts. The cationic antiseptic agent polyhexamethylene biguanide (PHMB) is effective in killing Acanthamoeba cysts and trophozoites by inhibiting membrane function.^3,19 In 1992 Larkin et al²⁰ reported its successful clinical use at a concentration of 0.02% in treatment of Acanthamoeba keratitis. Meni Care Plus storage solution for rigid gas permeable contact lenses contains 0.0005% PHMB. Even after a soaking time of 8 hours Meni Care Plus was ineffective against cysts of the species A lenticulata and A hatchetti at a concentration of 10⁵ cells/ml.

Cysts of Acanthamoeba are known to be highly resistant to extremes of pH and temperature and also to disinfectants of contact lens solutions.^3,21 Mazur et al²² demonstrated the viability and invasiveness of Acanthamoeba cysts after they had been stored in water at 4°C for a period of 24 years. These capabilities may vary between the different species and morphological groups. Our tests have shown that A lenticulata belonging to morphological group III showed the greatest resistance against the three tested disinfectant solutions. However, most clinical isolates are representatives of morphological group II and so far A lenticulata has not been reported to cause Acanthamoeba keratitis.^3,21

The main goal in prevention of Acanthamoeba keratitis is to keep the contact lens storage case free of micro-organisms, Acanthamoeba, and biofilm. Appropriate measures to prevent microbial contamination and the development of biofilm are frequent microwave treatment or boiling of the storage case (destroys Acanthamoeba cysts and trophozoites), mechanical cleansing and use of intensive cleaning solutions (removal of biofilm), avoidance of contact with contaminated water, frequent renewal of the storage case, use of effective solutions, and proper contact lens care.^{3,13,19,23–26} However, the development and use of storage solutions which are effective against Acanthamoeba is of great importance. We can recommend the Titmus H₂O₂ two step system for overnight soaking of contact lenses as an effective disinfectant against Acanthamoeba.