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International Journal of Applied Engineering Research

ISSN 0973-4562 Volume 9, Number 21(2014) pp. 8247-8254


Research India Publications
http://www.ripublication.com

Comparison of Contact-lens Clearing Solutions for


Acanthamoebial Infection in Vitro
Su-Youn Park1 and Suk-Yul Jung*2
1

Department of Clothing and Textiles, Konkuk University,


Seoul, 143-701, Republic of Korea
2
Department of Biomedical Laboratory Science, Molecular Diagnostics Research
Institute, Namseoul University, Cheonan, 331-707, Republic of Korea
E-mail: *syjung@nsu.ac.kr

Abstract
Acanthamoeba is a free-living protozoan pathogen which causes
blinding keratitis and fatal granulomatous encephalitis. A lot of contact
lens-clearing solutions are widely being used and they are applicable
for the washing of the contact lens or indirect eye washing due to easy
approaches and its inexpensive price. In this study, the effects of wellknown lens-clearing solutions to acanthamoebial infection were
analyzed in vitro using a human corneal epithelial cell (HCEC) as a
target cell. COMPLETE Multi-Purpose Solution Easy Rub Formula
had cytotoxicity of about 81.5% in its treatment of 100 ul to A.
castellanii. Its cytotoxicity was proportionally increased from 10 to 100
ul of its treatment. Fifty microliters of the lens-clearing solution
showed about 3.9 times more cytotoxicity than 10 ul treatment of A.
castellanii. One hundred ul of Cliwell had a cytotoxicity of about
54.7% and 61.5% to the host HCEC and A. castellanii, respectively.
Overall, as time increased, trophozoite forms of A. castellanii changed
into oval shapes. When treated with 90% of COMPLETE MultiPurpose Solution Easy Rub Formula and 10% of PYG, very little
number of the trophozoites was observed and they were not observed
after 24 h incubation. More than 50 % of Cliwell destroyed A.
castellanii trophozoites and changed the trophozoites into cysts of
smaller size. Taken together, COMPLETE Multi-Purpose Solution
Easy Rub Formula of the lens-clearing solution was more effective
than Cliwell to acanthamoebial infections. Moreover, it suggests that

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other chemicals should be analyzed for AK and other natural
compounds could be available to prevent antibiotic-resistance.
Keywords: Acanthamoeba castellanii; Contact-lens clearing solutions;
Cytotoxicity; Human corneal epithelial cell.

1. Introduction
Acanthamoeba is a free-living protozoan pathogen which causes blinding keratitis and
fatal granulomatous encephalitis. Acanthamoeba has been isolated from diverse
environments including air, soil, tap water, swimming pools and is known to be one of
the most ubiquitous protists [1-3]. In addition to their ability to produce human
infections, Acanthamoeba are shown to host bacterial pathogens and assist in their
environmental distribution and transmission to susceptible individuals [4, 5]. It
suggests that Acanthamoeba may also contribute indirectly to human infections caused
by pathogenic bacteria and acts as a trojan horse against nonpathogenic or pathogenic
bacteria. This concept is further strengthened with the finding that Acanthamoeba
resemble human macrophages in many ways, particularly in their phagocytic activity
and cell surface receptors [6], and that macrophages and Acanthamoeba exhibit
parallel mechanisms in their interactions with many bacterial pathogens.
Acanthamoeba infections are notoriously difficult to treat due to the rapid
propensity of the trophozoites to transform into cysts, which are highly resistant to
antimicrobial compounds [6, 7]. A. castellanii are causative agents of acanthamoebic
keratitis (AK) and is mostly associated with wearing contact lenses [4]. Because it is
similar in its symptoms with bacterial or viral keratitis, an incorrect diagnosis of AK
can be made. Thus, broad spectrums of antibiotics have been applied which would not
be enough to complete remove it. Its cytotoxicity has been analyzed using target cells,
such as, Chinese hamster ovary cells or human corneal epithelial cells. However, the
infectious pathogenesis and pathophysiology are not yet well understood. These
infections are of major concern in view of: (i) increasing numbers of
immunocompromised persons, (ii) increasing numbers of individuals undergoing
immunosuppressive therapy and excessive use of steroids, and (iii) global warming,
which may add to the ubiquity of these pathogens, and thus a possibility of increased
exposure to the susceptible hosts [7]. A lot of contact lens-clearing solutions have been
widely used and they are applicable for the washing of the contact lens or indirect eye
washing due to easy approaches and its inexpensive price. They include antibacterial
components and a washing liquid. Although companies advertise superiority of their
products, they have only compared them with two or three products from other
companies. In this study, the effects of well-known lens-clearing solutions to
acanthamoebial infection were analyzed in vitro using a human corneal epithelial cell
(HCEC) as a target cell.

Comparison of Contact-lens Clearing Solutions for Acanthamoebial

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2. Meterials and Methods


2.1 Culture of Acanthamoeba castellanii and human corneal epithelial cell
(HCEC)
A clinical isolate of A. castellanii belonging to T4 genotype, isolated from an amoebic
keratitis patient was used in the present study. A. castellanii was grown, without
shaking, in 12 ml of PYG medium [proteose peptone 0.75% (w/v), yeast extract 0.75%
(w/v) and glucose 1.5%(w/v)] in 75T tissue culture flasks at 30.
HCEC were routinely cultured as described by Araki-Sasaki et al. [8]. Briefly, HCEC
were grown in medium supplemented with penicillin (100 U/ml), streptomycin (100
mg/ml), 2mM glutamine and 10% fetal calf serum at 37 in a 5% CO2 incubator.
HCEC were co-cultured with A. castellanii in 96-well cell culture plates and then lensclearing solutions were directly treated.
2.2 Treatment of lens-clearing solutions to a target cell and A. castellanii
trophozoite
The lens-clearing solutions used were COMPLETE Multi-Purpose Solution Easy Rub
Formula (Abott Medical Optics, USA) and Cliwell (Bescon, Korea). HCEC were the
target cells. Growth rate of the trophozoites was also checked in vitro. After the target
cells were cultivated on 70% monolayer cell in a 75T flask, incomplete media for
washing was added and then EDTA. The 75T flask was treated to detach all cells for
20 min at 37. After removing the supernatant into a 15 ml tube, the tube was
centrifuged at 1,500 rpm for 5 min. These steps were performed three times. The
numbers of HCEC in the 15 ml tube were determined by a haemacytometer counting.
24 hours later, A. castellanii trophozoites (1105/well) were incubated for 30 min.
After half an hour, 5, 10, 50 and 100 of lens-clearing solution were added into a 96well plate. The trophozoites and HCEC were co-incubated for an additional 24 hr.
Finally, the number of amoeba and HCEC in the supernatant was determined by the
haemacytometer.
2.3 Microscopic analysis and cytotoxicity assay
Morphological findings were analyzed under a bright microscope. Analysis was based
on the destruction and membranous shrinkage of the trophozoites and destruction of
the HCEC. After the final incubation of HCEC, amoeba and lens-clearing solutions,
the analysis was done. For the cytotoxicity assay, after removing the supernatant,
EDTA was treated for 20 min. The number of living cells in the 96-wells was
determined by the hematocytometer. In particular, in this study, a traditional cell
counting method rather than biochemical approaches using lactate dehydrogenase was
applied to obtain more verifiable results. In fact, the survival of the cells was calculated
and cytotoxicity was predicted with a formula: 100%-(survival percent of
samples/survival percent of negative control without any contact-lens clearing
solutions) x 100%.

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Su-Youn Park & Suk-Yul Jung

3. Results
3.1 Analysis of in vitro cytotoxicity of contact-lens clearing solutions to A.
castellanii and HCEC
It was analyzed whether contact-lens clearing solutions was cytotoxic for A.
castellanii. A. castellanii and HCEC was co-cultured and then the contact-lens clearing
solutions were directly added into a 96-well cell culture plate. After 24 h of incubation,
their cytotoxicity was analyzed by a traditional cell counting method. At first, their
cytotoxicity assay was performed by releasing assay of lactate dehydrogenase (LDH).
Because the dilution of culture media could affect spectrophotometry, absorbance,
predicting the concentration of samples was not accurate and fluctuated severely.
Compared with a traditional cell counting assay and releasing assay of LDH, although
the cell counting assay was laborious and time-consuming, it was more effective and
had more accurate results (data not shown). As predicted in in vivo, HCEC was not
damaged by any contact-lens solutions. As a result, HCEC was not severely affected
by the lens-clearing solution of COMPLETE Multi-Purpose Solution Easy Rub
Formula (Fig. 1A). About 4.7% to 23.3% of cytotoxicity to HCEC was ignored in the
standard results. On the other hand, in the cytotoxicity by the cell counting assay,
COMPLETE Multi-Purpose Solution Easy Rub Formula had a cytotoxicity of about
81.5% in its treatment of 100 ul of A. castellanii (Fig. 1B). Its cytotoxicity was
proportionally increased from 10 to 100 ul when treated. Fifty microliter of the lensclearing solution showed about 3.9 times more cytotoxicity than 10 ul treatment of A.
castellanii (Fig. 1B).

Fig. 1: Cytotoxicity of lens-clearing solutions, e.g, COMPLETE Multi-Purpose


Solution Easy Rub Formula and Cliwell to HCEC and A. castellanii. A and B indicate
data of COMPLETE Multi-Purpose Solution Easy Rub Formula. C and D indicate data
of Cliwell. A and C indicate HCEC and B and D indicates A. castellanii. Bars show
standard deviation derived from three independent experiments.

Comparison of Contact-lens Clearing Solutions for Acanthamoebial

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Cliwell showed different patterns as compared with COMPLETE Multi-Purpose


Solution Easy Rub Formula (Fig. 1C and 1D). When treated with 100 ul, it had a
cytotoxicity of about 54.7% and 61.5% for the hosts HCEC and A. castellanii,
respectively. The amount of 100 ul was very toxic to both cells, especially the target
cell. Fifty microliters had a higher cytotoxicity for A. castellanii of about 4 times more
than a 10 ul treatment (Fig. 1D).
3.2 Microscopic analysis of A. castellanii by contact-lens clearing solutions
For more detailed analysis, microscopic findings were periodically performed up to 24
hr (Fig. 2). A. castellanii was cultured with PYG in vitro and then treated with lensclearing solutions. Overall, as time increased, trophozoite forms of A. castellanii
changed into oval shapes. Although relatively good culture conditions were provided
in 10% of COMPLETE Multi-Purpose Solution Easy Rub Formula for 24 hr, the
number of the trophozoites significantly decreased and their morphologies became
smaller, similar to the size of cysts, and transformed under harsh conditions (Fig. 2A).
On the other hand, under 90% of COMPLETE Multi-Purpose Solution Easy Rub
Formula and 10% of PYG, a very small number of the trophozoites was observed and
they were not observed after a 24 h incubation period (Fig. 2C).
Cliwell showed very similar data with cytotoxicity assays in Fig. 1 (Fig. 2D, 2E
and 2F). More than 50% of Cliwell destroyed A. castellanii trophozoites and changed
the trophozoites into cysts of smaller size (Fig. 2EB and 2F). After a 24 hr treatment of
90% Cliwell, no trophozoite or cyst was observed.

Fig. 2: Morphological findings of A. castellanii by lens-clearing solutions up to 24 h.


A, B and C indicate COMPLETE Multi-Purpose Solution Easy Rub Formula and D, E
and F indicate Cliwell. X200.

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4. DISCUSSION
A. castellanii is a single-celled protozoan that is widely distributed in the environment
and is well-known for causing human keratitis, a vision-threatening infection [9]. It is
well known that Acanthamoeba is a host for several bacteria, such as Escherichia coli,
Legionella pneumophila, Pseudomonas aeruginosa, Coxiella burnetii, Helicobacter
pylori, Vibro cholera [4]. Acanthamoeba can act as a Trojan horse, which is a harmful
property of bacteria, and is a food source of bacteria [4, 10].
A. castellanii is famous for causing AK to contact-lens wearers. A broad spectrum
of antibiotics and antimycotics has been recommended to remove bacteria, fungus and
other harmful materials. However, suitable therapeutic agents for AK have not been
well reported. For the prediction and development of therapeutic agents, in this study,
how much cytotoxic effects commercially available contact-lens solutions contained
and killed A. castellanii trophozoites were analyzed. Both solutions showed similar
cytotoxic function of about 40% in the treatment of 50 ul. However, 100 ul treatment
of COMPLETE Multi-Purpose Solution Easy Rub Formula showed about 20% higher
cytotoxic function than Cliwell. The main component of COMPLETE Multi-Purpose
Solution Easy Rub Formula is composed of polyhexamethylene biguanide
hydrochloride solution (PHMB). Cliwell consist of disodium edetate, sodium
phosphate and sodium chloride. It has been reported that PHMB has been given to an
AK patient [11]. The PHMB was successful with 0.2% treatment to remove
Acanthamoeba. On the other hand, it was reported that AK patient was resistant to
both PHMB-hexamidine and chlorhexidine-hexamidine treatment [12]. The patient
was a 39-year-old woman, who was recovering from a combinational therapy of the
above chemicals and keratoplasty. Disodium edetate is also known as EDTA sodium.
It can be used broadly as an anticoagulant. As compared with Cliwell, COMPLETE
Multi-Purpose Solution Easy Rub Formula contains PHMB which can be used with A.
castellanii. For the cytotoxicity assay, COMPLETE Multi-Purpose Solution Easy Rub
Formula was 20% more effective than Cliwell. In fact, its main components were only
advertised by the companies of two lens cleaners. If other components in the solutions
were reported, their effects would have been analyzed in detail. Therefore, this
suggests that other chemicals will be further investigated for AK, and other natural
compounds can prevent antibiotics-resistance.

5. Acknowledgments
Funding for this paper was provided by Namseoul University.

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