|Year : 2014 | Volume
| Issue : 2 | Page : 122-128
The efficacy of different commercial contact lens solutions on different concentrations of Acanthamoeba spp. trophozoites and cysts in Egypt
Yosra H Alam-Eldin, Heba A Aminou PhD
Department of Medical Parasitology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||03-Jul-2014|
|Date of Acceptance||15-Sep-2014|
|Date of Web Publication||19-Jan-2015|
Heba A Aminou
Department of Medical Parasitology, Faculty of Medicine, Ain Shams University, 11566 Cairo
Source of Support: None, Conflict of Interest: None
Acanthamoeba spp. keratitis is a devastating disease that can potentially result in threatening the sight of the affected eye. Ineffective lens-disinfecting systems and contaminated contact lens storage cases have been recognized as the main risk factors for the infection.
The present study aimed to evaluate the efficacy of nine different commercially available contact lens solutions in the Egyptian market against Acanthamoeba spp. trophozoites and 2-week-old cysts.
Materials and methods
Nine solutions were tested: eight multipurpose solutions (MPS) and one one-step hydrogen peroxide solution. Acanthamoeba spp. was isolated from a keratitic patient, cultivated on 1.5% non-nutrient agar (NNA), harvested, adjusted in two final concentrations of 5 × 10 3 and 5 × 10 5 trophozoites and cysts, and then incubated with the contact lens solution. The efficacy was tested at intervals of 2, 4, 6, 8, 10, and 24 h. Experiments were performed in triplicate. The viability was confirmed by reinoculation onto NNA seeded with Escherichia coli (NNA-E. coli).
Most of the tested solutions showed significant trophzoiticidal activity, whereas all of the tested solutions failed to eliminate the 2-week-old cysts completely. The one-step hydrogen peroxide system failed neutralization within the minimum manufacturer's disinfecting time as it killed all the cysts of 5 × 10 3 concentration after 10 h of soaking instead of 6 h; if used for this prolonged time, it could be hazardous to the users' eye. One of the MPS had high trophozoiticidal activity, but with an unknown recorded disinfectant, which could turn out to be of a toxic concentration or constitution.
Adjustment of the appropriate concentration of the disinfectant, the adequate exposure time, or even the development of new contact lens-disinfecting systems by manufacturers is needed to prevent Acanthamoeba spp. keratitis (AK). A MPS that fails to eradicate trophozoites or cysts within the minimum manufacturer's disinfecting time or one with an unknown recorded disinfectant should be avoided.
Keywords: Acanthamoeba spp. cysts, Acanthamoeba spp. keratitis, Acanthamoeba spp. trophozoites, contact lens solutions
|How to cite this article:|
Alam-Eldin YH, Aminou HA. The efficacy of different commercial contact lens solutions on different concentrations of Acanthamoeba spp. trophozoites and cysts in Egypt. Parasitol United J 2014;7:122-8
|How to cite this URL:|
Alam-Eldin YH, Aminou HA. The efficacy of different commercial contact lens solutions on different concentrations of Acanthamoeba spp. trophozoites and cysts in Egypt. Parasitol United J [serial online] 2014 [cited 2018 Mar 18];7:122-8. Available from: http://www.new.puj.eg.net/text.asp?2014/7/2/122/149564
| Introduction|| |
Acanthamoeba spp. are ubiquitous free-living protozoa most commonly found in freshwater and soil  . There are two stages in the life cycle of Acanthamoeba spp.: the motile trophozoite and the dormant cyst. Acanthamoeba spp. trophozoites usually feed on bacteria and yeast  . Encysted forms endure extreme environmental conditions of hyperosmolarity, glucose starvation, desiccation, extreme temperatures, and extreme pH ,, .
These protozoa are opportunistic causal agents of sight-threatening ulcerations of the cornea termed AK, disseminated infections (mostly cutaneous and nasopharyngeal), and usually fatal granulomatous amoebic encephalitis ,, .
This serious eye infection that primarily affects contact lens users  has increased drastically in recent years  . Ross et al.  found that 93.3% of the AK patients wore contact lenses. Poor hygiene practices such as failure to comply with recommended lens cleaning and disinfection procedures, and the rinsing and storing of lenses in nonsterile saline or tap water are recognized risk factors for infection  .
Acanthamoeba spp. attach to the surface of contact lenses and can be transmitted from the contact lens storage case onto the eye , . The infection is painful and is increasingly being recognized as a severe sight-threatening ocular infection worldwide  . Because diagnosis is difficult and often delayed, prevention seems to be the best solution for avoiding AK  .
Several types of soft contact lens disinfectant solutions are currently commercially available, including hydrogen peroxide-based solutions and MPS. This study attempted to investigate the amoebicidal activity of commercially locally marketed contact lens solutions against Acanthamoeba spp. trophozoites and 2-week-old cysts, aiming to evaluate their efficacy as disinfectant solutions.
| Materials and methods|| |
Type of study
Case control study.
Acanthamoeba isolation and culture
Corneal scrapings were collected from a keratitic patient attending the corneal outpatient clinic of the Research Institute of Ophthalmology, Giza, Egypt, in December. Acanthamoeba spp. isolation and testing were performed in the Diagnostic and Research Laboratory of Parasitic Diseases, Parasitology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt, during the period from December 2013 to April 2014.
Specimens were inoculated directly onto the surface of 1.5% NNA plates seeded with an Escherichia coli bacterial suspension and incubated in a humidified chamber at 30°C  . The presence of Acanthamoeba spp. could be seen by the clear tracks in the E. coli lawn on the NNA-E. coli surface produced by the feeding trophozoites of Acanthamoeba spp. Examination of the agar plate surface for the presence of amoebic growth was carried out daily for up to 7 days with light and inverted microscopes using a ×40 objective. Acanthamoeba spp. was identified by the specific morphology of the cyst and the trophozoite. Subcultures were prepared after 2 weeks from positive cultures with confirmed amoebic growth by cutting a small square of agar using a sterile scalpel and placing it upside down on new NNA-E. coli plates. The plates were incubated in humidified chambers at 30°C and examined after 24 h. Performing subculture several times facilitated the isolation of Acanthamoeba spp. Two-week-old Acanthamoeba spp. cysts were collected from 3-week cultures.
Preparation of Acanthamoeba spp. trophozoites and cysts
Agar surfaces were flooded with 5 ml of PBS and then scraped gently with an inoculating loop. Trophozoites/cysts were harvested from the suspension by centrifugation at 400 × g for 10 min. The supernatant was aspirated, and the sediment was washed twice in sterile PBS to eliminate most of the bacteria. Trophozoites/cysts in the resultant suspension were counted with a hemocytometer, and the suspension was standardized to contain 5 × 10 6 /ml  .
Commercial soft contact lens disinfectant solutions
All contact lens-disinfecting solutions tested were purchased from local retailers. Fresh solutions were removed from the original wrappings and used before their stated expiration date. They consisted of eight MPS and one one-step hydrogen peroxide system. All tested contact lens solutions' names, active disinfectant(s) [polyhexamethylene biguanide (PHMB), polyaminopropyl biguanide (PAPB)], disinfection time, other ingredient(s), contact lens type, and their manufacturers are listed in [Table 1].
The nine tested commercial contact lens solutions were designated T1 to T9, and the control experiment composed of PBS was designated C. Each experiment was performed in triplicate. Tests were performed in 15 ml centrifugation tubes. The calibrated 2-week-old trophozoite and cyst suspensions were added to 4 ml of the respective contact lens solution. Two test tubes were tested for the trophozoites (one calibrated to contain a final concentration of 5 × 10 3 and the other 5 × 10 5 ) and two test tubes for the cysts (one calibrated to contain a final concentration of 5 × 10 3 and the other 5 × 10 5 ).
After incubation in contact lens solutions at 37°C for 2, 4, 6, 8, 10, and 24 h, parasites adhered to the base of the centrifugation tubes were detached gently by a sterile cell scraper. This was followed by centrifugation at 500 ×g for 7 min. The deposit was agitated gently by pipetting up and down for 1 min  . Viability of the amoebae was assessed using 0.3% basic methylene blue. Unstained (viable) and stained (nonviable) parasites were enumerated in a hemocytometer, 10 min after stain addition, taking into consideration the dilution factor. For samples containing no viable cysts, an additional test was performed to confirm the results obtained. To evaluate the nonviability of all cysts and trophozoites, the suspension was reinoculated onto a NNA-E. coli plate, incubated at 30°C, and examined frequently for 2 weeks for the presence of trophozoites. Efficacies of the solutions were recorded as positive or negative , . As one single surviving cyst and trophozoite can give rise to a new 'amoeba population,' a contact lens solution was considered amoebicidal only if all trophozoites and cysts were eradicated in the given time.
| Results|| |
The nine contact lens disinfectant solutions examined in this study had different effects against trophozoite and cyst concentrations of 5 × 10 3 and 5 × 10 5 , as shown in [Table 2] for the trophozoites and [Table 3] for the cysts. The disinfectant effect of each lens solution was evaluated by inoculation on NNA-E. coli agar at the end of each time interval. The absence of viable trophozoites confirmed the disinfectant effect.
|Table 2 The viability of Acanthamoeba spp. trophozoites after lens solution exposures for 2, 4, 6, 8, 10, and 24 h|
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|Table 3 The viability of the 2-week-old Acanthamoeba spp. cysts after lens solution exposures for 2, 4, 6, 8, 10, and 24 h|
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Efficacy of contact lens disinfectant solutions against Acanthamoeba spp. trophozoites
Regarding experiments with a final concentration of 5 × 10 3 trophozoites, Opti-Free Express, Biotrue, Renu Multiplus, Contacid (MPSs), and Tutti (one-step hydrogen peroxide system) were able to destroy all Acanthamoeba spp. trophozoites within the manufacturers' minimum recommended disinfecting time (MMRDT). However, Perfect Care, Orion Multi, All In One Light and Fresh Look comfort failed to destroy all trophozoites within the MMRDT. Inoculation on NNA-E. coli media confirmed these results. Concerning the activity of tests against trophozoites of 5 × 10 5 concentration, all the tested commercial lens disinfectant solutions failed to eradicate Acanthamoeba spp. trophozoites within the MMRDT, except Opti-Free Express, Contacid, and Tutti.
Efficacy of contact lens disinfectant solutions against 2-week-old Acanthamoeba spp. cysts
Challenging the disinfectant solutions with 2-week-old Acanthamoeba spp. cysts with final concentrations of 5 × 10 3 and 5 × 10 5 showed the failure of all tested commercial contact lens solutions and control solutions to eradicate the cysts within the MMRDT. Results are shown in [Table 3].
| Discussion|| |
Acanthamoeba keratitis is a potentially devastating disease that, although rare, constantly presents difficulties in diagnosis and treatment  . Therefore, especially in contact lens users, prevention by proper and strict disinfection using effective contact lens solutions is of significant importance. However, the improper use of contact lens solutions may lead to lens contamination and thus predispose one to the development of AK. In the current study, eight commercial MPS and one one-step hydrogen peroxide system were challenged with Acanthamoeba spp. trophozoites and 2-week-old cysts.
The specimen was taken from corneal scraping and not collected from tap water, as tap water may contain several strains of Acanthamoeba spp. such as T 4 , T 5, and T 11  , whereas the majority of the human infections due to Acanthamoeba spp. have been associated with isolates of the T 4 genotype and more than 90% of AK cases have been linked with this genotype  .
With regard to the solutions' effectiveness against trophozoites at the concentration of 5 × 10 3 , our study demonstrated that the three solutions using PHMB (Perfect Care, All In One Light, Orion Multi) failed to eradicate Acanthamoeba spp. trophozoites within the MMRDT. However, Polat et al.  and Beattie et al.  reported the eradication of Acanthamoeba spp. trophozoites by All In One Light, within the recommended disinfecting time (4 h). The dissimilarity may be due to differences in the tested Acanthamoeba spp. strain or the experimental methodology.
Renu Multiplus, Fresh Look Comfort, and Biotrue, using PAPB as the disinfectant, varied in the eradication of the trophozoites within the MMRDT. Renu Multiplus was able to destroy the trophozoites within 2 h before the MMRDT, whereas Fresh Look Comfort required 8 h to eliminate the trophpozoites. Similarly, Beattie et al.  and Kobayashi et al.  found that Renu Multiplus is effective in the eradication of Acanthamoeba spp. trophozoites within the MMRDT. However, Polat et al.  reported that Renu Multiplus destroyed trophozoites of Acanthamoeba spp. after 6 h incubation time, but trophozoites were still viable before that. Padzik et al.  revealed that the strongest amoebostatic effect was visible after 24 h of exposure to Opti-Free and ReNu solution, and this is significantly longer than the MMRDT. A literature survey revealed that the current study is the first to evaluate the efficacy of Biotrue against Acanthamoeba spp. trophzoites and 2-week-old cysts, revealing its inefficiency in the eradication of the cysts up to 8 h for the 5 × 10 3 concentration and up to 24 h for the 5 × 10 5 concentration.
Opti-Free Express, which uses 0.001% polidronium chloride and 0.0005% myristamidopropyl dimethylamine, was able to destroy all trophozoites within 2 h before the MMRDT (6 h) at the concentration of 5 × 10 3 . In studies carried out by Beattie et al.  and Borazjani and Kilvington  , Opti-Free Express was considered trophozoiticidal within the MMRDT. Similarly, Ustüntürk and Zeybek  found that Opti-Free achieved total destruction of trophozoites before the MMRDT, but it had limited cysticidal activity. However, Mowrey-McKee and George  demonstrated that Opti-Free Express had a limited amoebicidal effect after the recommended disinfection time of 6 h.
The one-step hydrogen system, Tutti, used in our study was able to destroy the trophozoites after 2 h within the MMRDT (6 h). Previously, several one-step hydrogen peroxide solutions showed similar results , .
The present study demonstrated the different efficacies of the same disinfecting system when tested against the same concentration of trophozoites of the same strain. This is attributed to the different formulations of each commercial disinfecting solution, including buffering agents (such as sodium chloride), cleaning agents (such as sodium citrate), conditioning agents (such as tetronic), and surfactants (such as propylene glycol). Some of these inactive ingredients attenuate or potentiate the activity of the disinfectant. Different authors stated that the specific formulation of each product has been found to significantly affect its efficacy as a disinfectant ,, . For example, different MPS contain several types of nonionic surfactants (poloxamer, poloxamine, propylene glycol) that are reported to contribute to the survival of Acanthamoeba spp. because of their tendency to protect microorganisms by aiding biofilm formation and inducing amoebal encystment , . However, further studies are still needed to clarify individual and collective effects of these compounds on the general disinfectant efficacy.
Regarding the solutions' efficacy against trophozoites at the concentration of 5 × 10 5 , eradication occurred after 4 h in Tutti, after 6 h in Opti-Free Express and Renu Multiplus, after 10 h in Contacid, and after 24 h in all other commercial solutions. Therefore, all tested solutions failed to eradicate this higher concentration of trophozoites within the stated MMRDT, except Opti-Free Express, Contacid, and Tutti.
When commercial disinfecting systems were challenged against the 2-week-old cysts, all of them failed to destroy the resistant cysts in both concentrations within the MMRDT. Opti-Free Express, Renu Multiplus, Contacid, and Tutti eliminated the 5 × 10 3 cysts after 10 h. In contrast, complete eradication of this concentration of cysts occurred after 24 h with Biotrue. None of the tested disinfecting solutions were able to kill the 5 × 10 5 2-week-old cysts after 24 h of soaking. Comparable findings were reported by Polat et al.  who found that Renu Multiplus and All In One Light kill viable cysts after 12 h of soaking. Hiti et al.  observed that the one-step hydrogen peroxide system efficacy against cysts was variable depending on the tested strain and the solution concentration. In the present work, the one-step hydrogen peroxide system killed all the cysts of 5 × 10 3 concentration after 10 h of soaking and failed to kill all the cysts in the higher 5 × 10 5 concentration. This indicates that the neutralization of hydrogen peroxide in the Tutti system was not complete within the MMRDT and its effect extended to 10 h instead of 6 h, which may have a cytotoxic effect on the eyes of the users.
However, Kobayashi et al.  reported the failure of all solutions tested in their study against the 2-week-old cysts including the Renu Multiplus and the one-step hydrogen peroxide systems. This contradiction could be explained by the experiment design or the method of cyst production. Other recorded ways for cyst production are either by minimizing the E. coli content in the culture plate using moist heat (60°C) with a contact time of 60 min or by prolonged storage at room temperature for 24 months  . Beattie et al.  reported failure of Opti-Free Express, Renu Multiplus, and All In One Light to destroy cysts within the MMRDT. Similarly, Lakhundi et al.  found that none of the contact lens disinfection solutions used exhibited cysticidal effects.
Although Contacid exhibited significant effectiveness and killed all the viable trophozoites and the 5 × 10 3 cysts, its unknown active disinfectant renders it unsafe for use. The long MMRDT (8 h) may explain its effectiveness, but the higher concentration of disinfectant or its toxicity may be the reason for its efficacy as it contains boric acid which is a weak disinfectant agent.
Unfortunately, current ISO and FDA guidelines do not provide guidance for testing the efficacy of contact lens solutions against Acanthamoeba species. During their review of the methods used to evaluate the effectiveness of contact lens care solutions, Buck et al.  suggested that the apparent contradicting results on the effectiveness of contact lens solutions against Acanthamoeba spp. were partly due to differences in the organism strain and cyst production, preparation of the inoculum and neutralization of the test solution, recovery, the quantification method, and determination of the viability of survivors. The researchers concluded that such variables may be responsible for the dissimilarities in the results between studies.
A very critical point shown here is the lack of efficacy against Acanthamoeba spp. cysts of most tested solutions for soft contact lens storage, because most of the lens care solutions are routinely used for no more than a few minutes to overnight. Cysts were still viable after the overnight (8 h) exposure time. The question of whether complete eradication occurred after exposure to the tested solutions was an important issue. Surviving cysts in contact lens cases can excyst and multiply, initiating infection of the eye.
| Conclusion|| |
The main risk factor for corneal infection in contact lens wearers is the use of disinfecting systems ineffective in killing Acanthamoeba spp. cysts and trophozoites. All commercial solutions examined in this study proved not effective in eliminating Acanthamoeba spp. cysts within a reasonable time frame. Therefore, our results emphasize the necessity of an appropriate concentration of antiamoebic agents in contact lens-disinfecting solutions for it to be effective against Acanthamoeba spp. cysts and trophozoites. Another vital point shown is the need for adequate exposure time for effective killing of different stages of this parasite.
| Acknowledgements|| |
Both authors have participated in the literature search, the practical work, and manuscript preparation.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]