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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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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.
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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).
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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.
References
[1]
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