Accepted Manuscript Status of the effectiveness of contact lens disinfectants in Malaysia against keratitiscausing pathogens Farhat Abjani, Naveed Ahmed Khan, Suk Yul Jung, Ruqaiyyah Siddiqui PII:
S0014-4894(17)30279-5
DOI:
10.1016/j.exppara.2017.09.007
Reference:
YEXPR 7450
To appear in:
Experimental Parasitology
Received Date: 28 June 2017 Revised Date:
15 August 2017
Accepted Date: 11 September 2017
Please cite this article as: Abjani, F., Khan, N.A., Jung, S.Y., Siddiqui, R., Status of the effectiveness of contact lens disinfectants in Malaysia against keratitis-causing pathogens, Experimental Parasitology (2017), doi: 10.1016/j.exppara.2017.09.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Contact lens disinfectants marketed in Malaysia are ineffective against Acanthamoeba castellanii
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Status of the effectiveness of contact lens disinfectants in Malaysia against
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keratitis-causing pathogens
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Farhat Abjani,1 Naveed Ahmed Khan,1 Suk Yul Jung,2 Ruqaiyyah Siddiqui1*
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Department of Biological Sciences, School of Science and Technology, Sunway University,
Malaysia; 2Department of Biomedical Laboratory Science, Namseoul University, Korea.
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Short title: CLS and keratitis-causing pathogens
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*Corresponding address: Department of Biological Sciences, School of Science and
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Technology, Sunway University, Selangor, 47500, Malaysia. Tel: 60-(0)3-7491-8622. Ext:
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7172. Fax: 60-(0)3-5635-8630. E-mail: [email protected]
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Abstract The aim of this study was (i) to assess the antimicrobial effects of contact lens
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disinfecting solutions marketed in Malaysia against common bacterial eye pathogens and as
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well as eye parasite, Acanthamoeba castellanii, and (ii) to determine whether targeting cyst
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wall would improve the efficacy of contact lens disinfectants. Using ISO 14729 Stand-Alone
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Test for disinfecting solutions, bactericidal and amoebicidal assays of six different contact
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lens solutions including Oxysept®, AO SEPT PLUS, OPTI-FREE® pure moist®, Renu®
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freshTM , FreshKon® CLEAR and COMPLETE RevitaLensTM were performed using
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Manufacturers Minimum recommended disinfection time (MRDT). The efficacy of contact
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lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas
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aeruginosa, Methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes,
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Streptococcus pneumoniae, and Acanthamoeba castellanii. In addition, using chlorhexidine
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as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, we
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determined whether combination of both agents can enhance efficacy of marketed contact
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lens disinfectants against A. castellanii trophozoites and cysts, in vitro. The results revealed
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that all contact lens disinfectants tested showed potent bactericidal effects exhibiting 100%
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kill against all bacterial species tested. In contrast, none of the contact lens disinfectants had
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any effect on Acanthamoeba cysts viability. When tested against trophozoites, two
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disinfectants, Oxysept Multipurpose and AO-sept Multipurpose showed partial amoebicidal
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effects. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt
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cyst wall structure, the findings revealed that combination of both agents in contact lens
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disinfectants abolished viability of A. castellanii cysts and trophozoites. Given the inefficacy
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of contact lens disinfectants tested in this study, these findings present a significant concern
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to the public health. These findings revealed that targeting cyst wall by using cyst wall
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degrading molecules in contact lens disinfecting solutions will enhance their efficacy against
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this devastating eye infection.
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Key words: Acanthamoeba; Contact lens disinfectants; Bacteria; Eye Pathogens; Keratitis
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Introduction
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Microbial keratitis is a serious vision-threatening infection. Clinical features include redness, pain, tearing, blur vision and inflammation but symptoms vary depending on the
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causative agent (Ansari et al., 2013; Stapleton et al., 2007; 2012). It is caused by viral,
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bacterial, fungal, and protist pathogens. Contact lens use is one of the important risk factors
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in contracting microbial pathogens leading to microbial keratitis. This is of particular
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concerns for less developed countries where the use of contact lenses is on the rise. For
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example, recently we tested the effects of contact lens disinfectants marketed in Pakistan
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against microbial pathogens. The findings revealed that none of the commercially available
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contact lens disinfectants were effective against eye parasite, Acanthamoeba castellanii
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(Siddiqui et al., 2015). In part, this was attributed to the ability of Acanthamoeba parasite to
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transform into a phenotypically distinct cyst form under harsh conditions (Marciano-Cabral
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and Cabral, 2003; Visvesvara et al., 2007; Siddiqui and Khan, 2012). Cysts are double-
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walled, hardy structures with minimal metabolic activity making it resistant to harsh
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environmental conditions as well as biocides, resulting in prolonged treatment and often leads
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to infection recurrence Alizadeh et al., 2002; Turner et al., 2004; Khan, 2006). Given a
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plethora of contact lens disinfectants available in the market, it is important to assess their
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antimicrobial properties. Among a number of pathogens responsible for microbial keratitis,
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Oxysept®, AO SEPT PLUS, OPTI-FREE® pure moist®, Renu® freshTM , FreshKon® CLEAR
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and COMPLETE RevitaLensTM against common bacterial pathogens causing eye infections,
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including species of Pseudomonas, Staphylococcus, and Streptococcus, as well as leading eye
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parasite, Acanthamoeba castellanii. Moreover, as Acanthamoeba cyst walls are partly made
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of cellulose, we determined the inclusion of cellulase enzyme in contact lens disinfectants to
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degrade Acanthamoeba cyst walls in the improved efficacy of commercially available contact
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lens disinfectants.
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Material and Methods
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All chemicals were purchased from Sigma Labs (Poole, Dorset, England) unless otherwise stated. The stock solution of chlorhexidine (CHX) was prepared in methanol and
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was stored at room temperature. We used cellulase enzyme isolated from Trichoderma viride
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(70 Units) and chlorhexidine dihydrochloride (50 µM) in our experiments.
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Contact lens disinfecting solutions
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The type of contact lens disinfectants, the compositions and manufacturers’ Minimum Recommended Disinfection Time (MRDT) are listed in Table 1. All disinfectants were
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examined before the expiry date recommended by the Manufacturer. Six different contact
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lens disinfectants included, Oxysept®, AO SEPT PLUS, OPTI-FREE® pure moist®, Renu®
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freshTM, FreshKon® CLEAR and COMPLETE RevitaLensTM. These solutions were
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commonly marketed in Malaysia and were bought from nearby local pharmacy.
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Test organisms and growth conditions
Among bacteria, Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus
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aureus (MRSA), Streptococcus pyogenes, Streptococcus pneumoniae were tested in the
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present study. Methicillin-resistant Staphylococcus aureus is a clinical isolate from blood
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samples and was acquired from the Luton & Dunstable Hospital NHS Foundation Trust,
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Luton, England. Pseudomonas aeruginosa was isolated from a clinical sample at the Aga
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Khan University hospital. Streptococcus pneumoniae was isolated from the blood culture of a
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pneumonia patient. P. aeruginosa and MRSA were grown overnight in a shaking incubator
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by inoculating bacterial culture in an enriched media, Luria-Bertani (LB) broth at 37°C,
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whereas 5% CO2 was used to culture S. pneumoniae and S. pyogenes in brain–heart infusion
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(BHI) broth overnight at 37°C. Although ISO 14729 requires the use of ATCC strains of
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bacterial organisms for testing, the bacterial strains used in this study were clinical isolates
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derived from human specimens and relevant to test contact lens disinfectants. Acanthamoeba
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castellanii belonging to the T4 genotype (American Type Culture Collection, ATCC 50492)
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was used. It is originally isolated from a patient suffering from Acanthamoeba keratitis.
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Acanthamoeba castellanii was cultured in a T-75 tissue culture flask in 10 mL Proteose
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peptone-Yeast extract-Glucose (PYG) medium [0.75% (w/v) proteose peptone, 0.75% (w/v)
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yeast extract, and 1.5% (w/v) glucose] at 37°C as previously described (Sissons et al., 2005).
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Before performing assays, Fresh PYG media was added in each flask approximately 17-20 h
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before, to obtain amoebae in the trophozoite form. In order to obtain cysts, Non-nutrient agar
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(NNA) plates were seeded with trophozoites aseptically. Next, plates were kept at 30oC in an
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incubator for 14 days. Following this, 10 mL sterile PBS was added to each plate and cysts
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were scraped off by using a cell scraper. The cyst suspension was then centrifuged at 2,000 x
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g for 10 min. Finally, the resultant pellet was resuspended in PBS after discarding the
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supernatant and cysts were counted using a haemocytometer.
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Bactericidal assays Bactericidal assays were performed to evaluate the bactericidal activity of various contact lens disinfectants against MRSA, Pseudomonas aeruginosa, Streptococcus
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pneumoniae and Streptococcus pyogenes. International Organization for Standardization
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(ISO) 14729 Stand Alone Test criteria for examining contact lens disinfectants efficacy was
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employed as described previously (Rosenthal et al. 2002). Briefly, the optical density of
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bacterial broth cultures was adjusted to 0.22 at 595 nm using a spectrophotometer. Next, 106
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colony forming units (CFU) of bacterial cultures were added in Eppendorf tubes. Tubes were
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centrifuged at 12,000 x g for 2 min to pellet bacterial CFU. The supernatant was aspirated
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followed by addition of 200 µL of six different contact lens disinfectants to resuspend
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bacteria. Following 6 hours of incubation, bacteria were enumerated by plating as described
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previously (Abjani et al., 2016). Bacteria incubated with PBS alone and bacteria incubated
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with gentamicin 100 µg per mL were used as negative and positive controls. All experiments
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were performed several times in duplicate.
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Amoebicidal assays
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The amoebicidal assays were performed as previously described (Abjani et al., 2016).
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Briefly, contact lens disinfectants were incubated with 5 x 105 amoebae (final volume: 200
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µL) for 6 h at room temperature as per Manufacturers’ instructions. In some experiments,
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amoebae were incubated with contact lens disinfectants containing chlorhexidine (50 µM)
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and cellulase (70 units). Next, 0.1% Trypan blue was added to enumerate number of viable
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trophozoites by Trypan blue exclusion assay (dead cells having porous cell membrane allows
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Trypan blue entry and as a result cells appear blue whereas live cells remain unstained). To
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further confirm the viability of amoebae, amoebae were centrifuged at 2,000 x g for 5 min
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post-treatment to remove contact lens disinfectants, cellulase and residual drugs. The
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for 72 h at 30°C. Additionally, amoebae trophozoites were inoculated in Roswell Park
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Memorial Institute-1640 (RPMI) medium alone as a control. All experiments were conducted
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several times in duplicate. The data are presented as mean ± standard error.
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Cysticidal assays
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Cysticidal assays were performed as described above. Briefly, cysts (5 x 104 cyst) were incubated with contact lens disinfectants, along with chlorhexidine (50 µM) and
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cellulase (70 units) for 6 h at room temperature as above, followed by Trypan blue exclusion
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assay and enumeration of dead cells using haemocytometer counting. The viability of
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amoebae cysts was further confirmed using inoculating cysts, post-treatment, in fresh PYG as
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described above in 96-well plates for 72 h at 30°C. For negative control, cysts were
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inoculated in Roswell Park Memorial Institute-1640 (RPMI) medium alone. All experiments
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were conducted several times in duplicate. The data are presented as mean ± standard error.
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Results
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Bactericidal effect of multipurpose contact lens disinfectant against P. aeruginosa and
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MRSA, Streptococcus pyogenes and Streptococcus pneumoniae To evaluate the bactericidal effects of contact lens disinfectants on P. aeruginosa and
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MRSA, assays were performed by inoculating 106 bacteria in 200 µL of contact lens
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disinfectants. The results revealed that all six contact lens disinfectants: Oxysept®, AO SEPT
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PLUS, OPTI-FREE® PureMoist, COMPLETE RevitaLens, Renu® fresh and FreshKon®
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CLEAR exhibited potent bactericidal effects against MRSA (Fig. 1A), P. aeruginosa (Fig.
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1B), Streptococcus pyogenes (Fig. 1C), and Streptococcus pneumoniae (Fig. 1D) within
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Manufacturer’s Minimum Recommended Disinfection time (MRDT) and no bacterial
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colonies were observed. For positive controls, bacteria incubated with 100 µg per mL of 7
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gentamicin abolished bacterial viability. When incubated alone, bacteria exhibited growth
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compared with the original inoculum.
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Combination of cellulase and chlorhexidine improved efficacy of contact lens disinfectants
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against A. castellanii trophozoites
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To determine the effects of contact lens disinfectants against Acanthamoeba
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castellanii trophozoites, amoebae were incubated contact lens solutions. The results showed
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that all four multipurpose Contact lens disinfectant (without hydrogen peroxide) OPTI-
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FREE® pure moist®, Renu® freshTM, FreshKon® CLEAR and COMPLETE RevitaLensTM
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were ineffective in killing A. castellanii trophozoites. There was no difference between the
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original inoculum (5 x 105 amoebae) and the number of viable Acanthamoeba post-treatment,
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4.29 x 105 ± 3.55 x 104, 4.07 x 105 ± 7.3 x 104, 4.07 x 105 ± 7.7 x 104 and 3.67 x 105 ± 2.0 x
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103 using OPTI-FREE® pure moist®, Renu® freshTM, FreshKon® CLEAR and COMPLETE
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RevitaLensTM respectively (Fig. 2). Moreover, when amoebae were inoculated in fresh PYG
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post-treatment, viable trophozoites emerged within 24 h. In contrast, hydrogen peroxide
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containing disinfectants showed partial amoebicidal effects and the number of viable amoeba
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was 7.35 x 104 ± 3.55 x 104, 4.95 x 104 ± 5.5 x 103 for Oxysept®, and AO SEPT PLUS
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respectively (Fig. 2).
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To determine whether the addition of cellulase and chlorhexidine at micromolar
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concentration improve the efficacy of contact lens disinfectants, cellulase enzyme was added
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at 70 µM. The results revealed that the addition of cellulase showed no effect on the viability
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of trophozoites and after six hours of incubation the number of viable amoebae was 3.28 x
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105 ± 3.5 x 103. When cellulase was combined with contact lens disinfectants, OPTI-FREE®
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pure moist®, Renu® freshTM, FreshKon® CLEAR and COMPLETE RevitaLensTM the number
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of viable amoeba was 2 x 105 ± 2 x 103, 2.68 x 105 ± 4.0 x 104, 2.67 x 105 ± 1.1 x 104 and
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2.66 x 105 ± 1.5 x 104 respectively but upon re-inoculation in fresh PYG, viable trophozoites
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emerged within 72 h. Similarly, chlorhexidine alone (up to 50µM) showed potent
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amoebicidal effects on the viability of A. castellanii trophozoites. Notably, contact lens disinfectants, OPTI-FREE® pure moist®, Renu® freshTM ,
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FreshKon® CLEAR and COMPLETE RevitaLensTM in combination with cellulase (70 units)
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and chlorhexidine (50µM) abolished viability of A. castellanii trophozoites within 6 h of
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incubation and the number was reduced to 3.85 x 104 ± 1.2 x 104, 1.50 x 104 ± 4.00 x 103,
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3.10 x 104 ± 9 x 103 and 3 x 104 ± 1 x 103 respectively (Fig. 2). Post-treatment, amoebae were
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unable to revive in fresh PYG medium even after 72 h (data not shown). Amoebae in RPMI
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as negative control and in methanol as solvent control for chlorhexidine showed no
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amoebicidal effect and the number was 5.52 x 105 ± 6.5 x 104 and 4.01 x 105 ± 5.3 x 104
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respectively.
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Addition of cellulase and chlorhexidine improved efficacy of contact lens disinfectants
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against cysts of A. castellanii
Cysticidal assays were performed to evaluate the efficacy of contact lens
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disinfectants. The results revealed that all four multipurpose Contact lens disinfectant
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(without hydrogen peroxide) OPTI-FREE® pure moist®, Renu® freshTM, FreshKon® CLEAR
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and COMPLETE RevitaLensTM were ineffective in killing A. castellanii cyst (Fig. 3), and
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viable amoebae emerged when inoculated in fresh PYG compared with the original inoculum
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(dotted line). Post-treatment, viable trophozoites emerged within 72 h. Hydrogen peroxide
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containing disinfectants (Oxysept® and AO SEPT PLUS) showed partial cysticidal effects
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(Fig. 3). Cellulase enzyme when used at 70µM showed no significant effect and the number 9
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was 1.72 x 105 ± 1.69 x 104. However, combination of cellulase and chlorhexidine with
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contact lens disinfectants abolished viability of A. castellanii cysts and amoebae were unable
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to revive when inoculated in fresh PYG (Fig. 3).
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Discussion
Keratitis is a vision-threatening disease of the eye often associated with the use of contact lens (Mascarenhas et al., 2015). If not treated promptly, it can lead to permanent
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vision impairment. Improper contact lens hygiene, use of homemade saline solutions for
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cleaning and storing lenses as well as using expired disinfectants further leads to an increase
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risk of microbial keratitis among contact lens wearers (Stapleton et al., 2007; 2012; Lorenzo-
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Morales et al., 2013). In this study, we determine the effectiveness of contact lens
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disinfectants available commercially in Malaysia against common bacterial eye pathogens
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and corneal parasite, Acanthamoeba castellanii. Our finding revealed that all contact lens
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disinfectants tested exhibited potent bactericidal effects against species tested. However, none
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of the contact lens disinfectants completely destroyed A. castellanii trophozoites and cyst.
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These findings are consistent with several recent studies indicating that marketed contact lens
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disinfectants are ineffective in completely destroying amoebae (Kolar et al., 2015; Padzik et
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al., 2014; Polat et al., 2007; Ustüntürk and Zeybek, 2014). In particular, there show limited
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efficacy against cysts. Chlorhexidine at 50 µM concentration destroyed amoeba within 6 h of
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incubation time at room temperature. However, cellulase treatment had no effect on the
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viability of amoebae and parasites re-emerged as viable trophozoites upon re-inoculation in
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PYG. In contrast, a combination of chlorhexidine and cellulase together with contact lens
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disinfectants proved highly effective in killing both A. castellanii cyst and trophozoites. Our
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findings showed that targeting the cellulose synthesis pathway inhibits Acanthamoeba
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switching into the cyst form. This is likely due to the ability of cellulase to destroy cyst walls
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ultimately leading to its death. This is consistent with our previous findings which showed
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that the addition of cellulase enzyme together with chlorhexidine enhanced the efficacy of
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contact lens solutions marketed in Pakistan (Abjani et al., 2016). As cellulose synthesis is
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limited to plants, yeast, and some bacteria, it is likely to have minimal side effects against
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human cells. Overall, the results obtained from our previous as well as present study clearly
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demonstrate that there is a need for improved contact lens disinfectant against Acanthamoeba
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castellanii. As cyst are partly made of cellulose, degradation of cellulose by cellulase enzyme
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and chlorhexidine is a promising strategy in targeting A. castellanii trophozoites as well as
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cysts. Our findings also highlight the ineffectiveness of commercially available contact lens
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disinfectant in Malaysia that present a major health risk to the public. We propose that similar
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strategy can be employed in the improved treatment of Acanthamoeba keratitis that can
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normally last up to a year and often complicated due to infection recurrence (Clarke et al.,
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2012), however intensive research is needed over the next few years to determine the
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translational value of these findings.
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Conflicts of interest: None to declare.
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Acknowledgments: The authors are grateful for the kind support provided by Sunway
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University, Malaysia and Lancaster University, UK.
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Figure Legends
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Figure 1. Commonly used contact lens disinfectants marketed in Malaysia were tested
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including Oxysept (OS), AO SEPT PLUS (AO SP), OPTI-FREE pure moist (OF PM), Renu
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fresh (RF), FreshKon CLEAR (FKC) and COMPLETE RevitaLens (CRL) against MRSA
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(A), P. aeruginosa (B), Streptococcus pyogenes (C), and Streptococcus pneumoniae (D)
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using Manufacturer’s Minimum Recommended Disinfection time (MRDT) as described in
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Materials and Methods. 100 µg/ml of gentamicin was used as a positive control, while
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bacteria incubated in PBS alone were used as a negative control. All six disinfectants tested
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abolished bacterial viability. The data is representative of several experiments performed in
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duplicate as expressed as the mean ± standard error.
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Figure 2. Contact lens disinfectants including Oxysept (OS), AO SEPT PLUS (AO SP),
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OPTI-FREE pure moist (OF PM), Renu fresh (RF), FreshKon CLEAR (FKC) and
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COMPLETE RevitaLens (CRL) were tested against A. castellanii trophozoites as described
335
in Materials and Methods. Note that none of the contact lens disinfectants destroyed amoebae
14
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effectively. However, addition of chlorhexidine (CHX) and cellulase to contact lens
337
disinfectant abolished A. castellanii viability as compared to the contact lens disinfectant
338
alone. The dotted line represents initial inoculum of trophozoites. The data is expressed as the
339
mean ± standard error of several experiments performed in duplicate.
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Figure 3. Contact lens disinfectants including Oxysept (OS), AO SEPT PLUS (AO SP),
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OPTI-FREE pure moist (OF PM), Renu fresh (RF), FreshKon CLEAR (FKC) and
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COMPLETE RevitaLens (CRL) were tested against A. castellanii cysts as described in
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Materials and Methods. Note that none of the contact lens disinfectants destroyed amoebae
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cysts. However, addition of chlorhexidine (CHX) and cellulase to contact lens disinfectant
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abolished A. castellanii viability as compared to the contact lens disinfectant alone. The
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dotted line represents initial inoculum of cysts. The data is expressed as the mean ± standard
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error of several experiments performed in duplicate.
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ACCEPTED MANUSCRIPT Table 1 List of contact lens disinfectants tested in the present study together with their ingredients, manufacturer, type of solution and Minimum recommended disinfection time (MRDT). Ingredients
Manufacturer
Oxysept®
3% Hydrogen peroxide
Abbot Medical Optics, Hangzhou
AO SEPT PLUS
3% Hydrogen peroxide
Alcon laboratories, Inc. Fort Worth, USA.
6h
Hydrogen peroxide system
OPTI-FREE® PureMoist®
POLYQUAD® (polyquaternium-1) 0.001% and ALDOX® (Myristamidopropyl dimethylamine) 0.0006% preservatives
Alcon laboratories, Inc. Fort Worth, Texas USA.
6h
Multi-purpose Disinfecting solution
COMPLETE RevitaLensTM
Alexidine dihydrochloride 0.00016%, Polyquaternium-1 0.0003%, boric acid, sodium borate decahydrate, TETRONIC 904, edetate disodium, sodium citrate, sodium chloride and purified water.
Abbot Medical Optics, Hangzhou
6h
Multi-purpose Disinfecting solution
Renu® freshTM
HYDRANATE Baush & Lomb (hydroxyalkylphosphonate), boric Greenville, acid, edetate disodium, poloxamine, USA. sodium borate and sodiumchloride, preserved with DYMEDTM (polyaminopropyl biguanide) 0.0001%
4h
Multi-purpose solution
Sodium and Potassium chloride, Disodium Edetate, Polyhexanide, Poloxamer, Hypromellose and Sodium Phosphate Buffer
6h
Multi-purpose solution
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FreshKon® CLEAR
Minimum Type of recommended solution disinfection time (MRDT) 6h Hydrogen peroxide system
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Solution
Oculus private limited, Singapore
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Fig. 1A
1.80E+09
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1.60E+09
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2.00E+08 0.00E+00 PBS
Gentamycin
MRSA
Renu fesh
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6.00E+08
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MRSA (CFU/ml)
1.40E+09
FreshKon CLEAR
OPTI-FREE pure moist
COMPLETE RevitaLens
MRSA + Contact Lens Disinfectant
Oxysept
AO SEPT PLUS
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Fig. 1B
3.50E+09
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5.00E+08 0.00E+00 PBS
Gentamycin
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Renu fesh
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P. aeruginosa (CFU/ml)
3.00E+09
P. aeruginosa
FreshKon CLEAR
OPTI-FREE pure moist
COMPLETE RevitaLens
Oxysept
P. aeruginosa + Contact Lens Disinfectant
AO SEPT PLUS
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Fig. 1C
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1.40E+09
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2.00E+08 0.00E+00 PBS
Gentamycin
S. pyogenes
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4.00E+08
Renu fesh
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S. pyogenes (CFU/ml)
1.20E+09
FreshKon CLEAR
OPTI-FREE pure moist
COMPLETE RevitaLens
S. pyogenes + Contact Lens Disinfectant
Oxysept
AO SEPT PLUS
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Fig. 1D
3.00E+09
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1.00E+09
0.00E+00 PBS
Gentamycin
Renu fesh
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5.00E+08
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S. Pneumoniae (CFU/ml)
2.50E+09
S. pneumoniae
FreshKon CLEAR
OPTI-FREE pure moist
COMPLETE RevitaLens
Oxysept
S. pneumoniae + Contact Lens Disinfectant
AO SEPT PLUS
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Fig. 2
P < 0.05
P < 0.05
5.00E+05 P < 0.05
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2.00E+05 1.00E+05
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P < 0.05
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Number of viable Acanthamoeba
6.00E+05
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7.00E+05
ACCEPTED MANUSCRIPT
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Fig. 3
SC
2.50E+05
P < 0.05
P < 0.05 P < 0.05 P < 0.05
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1.50E+05
0.00E+00
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1.00E+05
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Number of Acanthamoeba.castellanii
3.00E+05
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Highlights Efficacy of contact lens solutions was determined against keratitis-causing microbes.
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Of the six contact lens solutions tested, all were effective in killing common bacterial eye pathogens.
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None of them showed any cysticidal effects.
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Addition of cellulase and chlorhexidine improved efficacy of CL solutions
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S0014-4894(17)30279-5
DOI:
10.1016/j.exppara.2017.09.007
Reference:
YEXPR 7450
To appear in:
Experimental Parasitology
Received Date: 28 June 2017 Revised Date:
15 August 2017
Accepted Date: 11 September 2017
Please cite this article as: Abjani, F., Khan, N.A., Jung, S.Y., Siddiqui, R., Status of the effectiveness of contact lens disinfectants in Malaysia against keratitis-causing pathogens, Experimental Parasitology (2017), doi: 10.1016/j.exppara.2017.09.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Contact lens disinfectants marketed in Malaysia are ineffective against Acanthamoeba castellanii
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Status of the effectiveness of contact lens disinfectants in Malaysia against
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keratitis-causing pathogens
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Farhat Abjani,1 Naveed Ahmed Khan,1 Suk Yul Jung,2 Ruqaiyyah Siddiqui1*
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Department of Biological Sciences, School of Science and Technology, Sunway University,
Malaysia; 2Department of Biomedical Laboratory Science, Namseoul University, Korea.
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Short title: CLS and keratitis-causing pathogens
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*Corresponding address: Department of Biological Sciences, School of Science and
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Technology, Sunway University, Selangor, 47500, Malaysia. Tel: 60-(0)3-7491-8622. Ext:
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7172. Fax: 60-(0)3-5635-8630. E-mail: [email protected]
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Abstract The aim of this study was (i) to assess the antimicrobial effects of contact lens
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disinfecting solutions marketed in Malaysia against common bacterial eye pathogens and as
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well as eye parasite, Acanthamoeba castellanii, and (ii) to determine whether targeting cyst
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wall would improve the efficacy of contact lens disinfectants. Using ISO 14729 Stand-Alone
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Test for disinfecting solutions, bactericidal and amoebicidal assays of six different contact
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lens solutions including Oxysept®, AO SEPT PLUS, OPTI-FREE® pure moist®, Renu®
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freshTM , FreshKon® CLEAR and COMPLETE RevitaLensTM were performed using
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Manufacturers Minimum recommended disinfection time (MRDT). The efficacy of contact
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lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas
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aeruginosa, Methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes,
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Streptococcus pneumoniae, and Acanthamoeba castellanii. In addition, using chlorhexidine
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as an antiamoebic compound and cellulase enzyme to disrupt cyst wall structure, we
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determined whether combination of both agents can enhance efficacy of marketed contact
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lens disinfectants against A. castellanii trophozoites and cysts, in vitro. The results revealed
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that all contact lens disinfectants tested showed potent bactericidal effects exhibiting 100%
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kill against all bacterial species tested. In contrast, none of the contact lens disinfectants had
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any effect on Acanthamoeba cysts viability. When tested against trophozoites, two
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disinfectants, Oxysept Multipurpose and AO-sept Multipurpose showed partial amoebicidal
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effects. Using chlorhexidine as an antiamoebic compound and cellulase enzyme to disrupt
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cyst wall structure, the findings revealed that combination of both agents in contact lens
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disinfectants abolished viability of A. castellanii cysts and trophozoites. Given the inefficacy
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of contact lens disinfectants tested in this study, these findings present a significant concern
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to the public health. These findings revealed that targeting cyst wall by using cyst wall
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degrading molecules in contact lens disinfecting solutions will enhance their efficacy against
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this devastating eye infection.
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Key words: Acanthamoeba; Contact lens disinfectants; Bacteria; Eye Pathogens; Keratitis
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Introduction
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Microbial keratitis is a serious vision-threatening infection. Clinical features include redness, pain, tearing, blur vision and inflammation but symptoms vary depending on the
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causative agent (Ansari et al., 2013; Stapleton et al., 2007; 2012). It is caused by viral,
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bacterial, fungal, and protist pathogens. Contact lens use is one of the important risk factors
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in contracting microbial pathogens leading to microbial keratitis. This is of particular
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concerns for less developed countries where the use of contact lenses is on the rise. For
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example, recently we tested the effects of contact lens disinfectants marketed in Pakistan
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against microbial pathogens. The findings revealed that none of the commercially available
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contact lens disinfectants were effective against eye parasite, Acanthamoeba castellanii
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(Siddiqui et al., 2015). In part, this was attributed to the ability of Acanthamoeba parasite to
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transform into a phenotypically distinct cyst form under harsh conditions (Marciano-Cabral
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and Cabral, 2003; Visvesvara et al., 2007; Siddiqui and Khan, 2012). Cysts are double-
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walled, hardy structures with minimal metabolic activity making it resistant to harsh
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environmental conditions as well as biocides, resulting in prolonged treatment and often leads
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to infection recurrence Alizadeh et al., 2002; Turner et al., 2004; Khan, 2006). Given a
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plethora of contact lens disinfectants available in the market, it is important to assess their
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antimicrobial properties. Among a number of pathogens responsible for microbial keratitis,
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Oxysept®, AO SEPT PLUS, OPTI-FREE® pure moist®, Renu® freshTM , FreshKon® CLEAR
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and COMPLETE RevitaLensTM against common bacterial pathogens causing eye infections,
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including species of Pseudomonas, Staphylococcus, and Streptococcus, as well as leading eye
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parasite, Acanthamoeba castellanii. Moreover, as Acanthamoeba cyst walls are partly made
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of cellulose, we determined the inclusion of cellulase enzyme in contact lens disinfectants to
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degrade Acanthamoeba cyst walls in the improved efficacy of commercially available contact
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lens disinfectants.
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Material and Methods
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All chemicals were purchased from Sigma Labs (Poole, Dorset, England) unless otherwise stated. The stock solution of chlorhexidine (CHX) was prepared in methanol and
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was stored at room temperature. We used cellulase enzyme isolated from Trichoderma viride
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(70 Units) and chlorhexidine dihydrochloride (50 µM) in our experiments.
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Contact lens disinfecting solutions
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The type of contact lens disinfectants, the compositions and manufacturers’ Minimum Recommended Disinfection Time (MRDT) are listed in Table 1. All disinfectants were
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examined before the expiry date recommended by the Manufacturer. Six different contact
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lens disinfectants included, Oxysept®, AO SEPT PLUS, OPTI-FREE® pure moist®, Renu®
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freshTM, FreshKon® CLEAR and COMPLETE RevitaLensTM. These solutions were
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commonly marketed in Malaysia and were bought from nearby local pharmacy.
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Test organisms and growth conditions
Among bacteria, Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus
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aureus (MRSA), Streptococcus pyogenes, Streptococcus pneumoniae were tested in the
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present study. Methicillin-resistant Staphylococcus aureus is a clinical isolate from blood
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samples and was acquired from the Luton & Dunstable Hospital NHS Foundation Trust,
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Luton, England. Pseudomonas aeruginosa was isolated from a clinical sample at the Aga
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Khan University hospital. Streptococcus pneumoniae was isolated from the blood culture of a
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pneumonia patient. P. aeruginosa and MRSA were grown overnight in a shaking incubator
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by inoculating bacterial culture in an enriched media, Luria-Bertani (LB) broth at 37°C,
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whereas 5% CO2 was used to culture S. pneumoniae and S. pyogenes in brain–heart infusion
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(BHI) broth overnight at 37°C. Although ISO 14729 requires the use of ATCC strains of
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bacterial organisms for testing, the bacterial strains used in this study were clinical isolates
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derived from human specimens and relevant to test contact lens disinfectants. Acanthamoeba
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castellanii belonging to the T4 genotype (American Type Culture Collection, ATCC 50492)
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was used. It is originally isolated from a patient suffering from Acanthamoeba keratitis.
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Acanthamoeba castellanii was cultured in a T-75 tissue culture flask in 10 mL Proteose
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peptone-Yeast extract-Glucose (PYG) medium [0.75% (w/v) proteose peptone, 0.75% (w/v)
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yeast extract, and 1.5% (w/v) glucose] at 37°C as previously described (Sissons et al., 2005).
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Before performing assays, Fresh PYG media was added in each flask approximately 17-20 h
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before, to obtain amoebae in the trophozoite form. In order to obtain cysts, Non-nutrient agar
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(NNA) plates were seeded with trophozoites aseptically. Next, plates were kept at 30oC in an
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incubator for 14 days. Following this, 10 mL sterile PBS was added to each plate and cysts
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were scraped off by using a cell scraper. The cyst suspension was then centrifuged at 2,000 x
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g for 10 min. Finally, the resultant pellet was resuspended in PBS after discarding the
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supernatant and cysts were counted using a haemocytometer.
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Bactericidal assays Bactericidal assays were performed to evaluate the bactericidal activity of various contact lens disinfectants against MRSA, Pseudomonas aeruginosa, Streptococcus
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pneumoniae and Streptococcus pyogenes. International Organization for Standardization
125
(ISO) 14729 Stand Alone Test criteria for examining contact lens disinfectants efficacy was
126
employed as described previously (Rosenthal et al. 2002). Briefly, the optical density of
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bacterial broth cultures was adjusted to 0.22 at 595 nm using a spectrophotometer. Next, 106
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colony forming units (CFU) of bacterial cultures were added in Eppendorf tubes. Tubes were
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centrifuged at 12,000 x g for 2 min to pellet bacterial CFU. The supernatant was aspirated
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followed by addition of 200 µL of six different contact lens disinfectants to resuspend
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bacteria. Following 6 hours of incubation, bacteria were enumerated by plating as described
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previously (Abjani et al., 2016). Bacteria incubated with PBS alone and bacteria incubated
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with gentamicin 100 µg per mL were used as negative and positive controls. All experiments
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were performed several times in duplicate.
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Amoebicidal assays
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The amoebicidal assays were performed as previously described (Abjani et al., 2016).
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Briefly, contact lens disinfectants were incubated with 5 x 105 amoebae (final volume: 200
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µL) for 6 h at room temperature as per Manufacturers’ instructions. In some experiments,
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amoebae were incubated with contact lens disinfectants containing chlorhexidine (50 µM)
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and cellulase (70 units). Next, 0.1% Trypan blue was added to enumerate number of viable
141
trophozoites by Trypan blue exclusion assay (dead cells having porous cell membrane allows
142
Trypan blue entry and as a result cells appear blue whereas live cells remain unstained). To
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further confirm the viability of amoebae, amoebae were centrifuged at 2,000 x g for 5 min
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post-treatment to remove contact lens disinfectants, cellulase and residual drugs. The
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146
for 72 h at 30°C. Additionally, amoebae trophozoites were inoculated in Roswell Park
147
Memorial Institute-1640 (RPMI) medium alone as a control. All experiments were conducted
148
several times in duplicate. The data are presented as mean ± standard error.
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Cysticidal assays
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Cysticidal assays were performed as described above. Briefly, cysts (5 x 104 cyst) were incubated with contact lens disinfectants, along with chlorhexidine (50 µM) and
152
cellulase (70 units) for 6 h at room temperature as above, followed by Trypan blue exclusion
153
assay and enumeration of dead cells using haemocytometer counting. The viability of
154
amoebae cysts was further confirmed using inoculating cysts, post-treatment, in fresh PYG as
155
described above in 96-well plates for 72 h at 30°C. For negative control, cysts were
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inoculated in Roswell Park Memorial Institute-1640 (RPMI) medium alone. All experiments
157
were conducted several times in duplicate. The data are presented as mean ± standard error.
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Results
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Bactericidal effect of multipurpose contact lens disinfectant against P. aeruginosa and
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MRSA, Streptococcus pyogenes and Streptococcus pneumoniae To evaluate the bactericidal effects of contact lens disinfectants on P. aeruginosa and
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MRSA, assays were performed by inoculating 106 bacteria in 200 µL of contact lens
164
disinfectants. The results revealed that all six contact lens disinfectants: Oxysept®, AO SEPT
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PLUS, OPTI-FREE® PureMoist, COMPLETE RevitaLens, Renu® fresh and FreshKon®
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CLEAR exhibited potent bactericidal effects against MRSA (Fig. 1A), P. aeruginosa (Fig.
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1B), Streptococcus pyogenes (Fig. 1C), and Streptococcus pneumoniae (Fig. 1D) within
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Manufacturer’s Minimum Recommended Disinfection time (MRDT) and no bacterial
169
colonies were observed. For positive controls, bacteria incubated with 100 µg per mL of 7
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gentamicin abolished bacterial viability. When incubated alone, bacteria exhibited growth
171
compared with the original inoculum.
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Combination of cellulase and chlorhexidine improved efficacy of contact lens disinfectants
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against A. castellanii trophozoites
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To determine the effects of contact lens disinfectants against Acanthamoeba
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castellanii trophozoites, amoebae were incubated contact lens solutions. The results showed
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that all four multipurpose Contact lens disinfectant (without hydrogen peroxide) OPTI-
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FREE® pure moist®, Renu® freshTM, FreshKon® CLEAR and COMPLETE RevitaLensTM
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were ineffective in killing A. castellanii trophozoites. There was no difference between the
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original inoculum (5 x 105 amoebae) and the number of viable Acanthamoeba post-treatment,
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4.29 x 105 ± 3.55 x 104, 4.07 x 105 ± 7.3 x 104, 4.07 x 105 ± 7.7 x 104 and 3.67 x 105 ± 2.0 x
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103 using OPTI-FREE® pure moist®, Renu® freshTM, FreshKon® CLEAR and COMPLETE
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RevitaLensTM respectively (Fig. 2). Moreover, when amoebae were inoculated in fresh PYG
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post-treatment, viable trophozoites emerged within 24 h. In contrast, hydrogen peroxide
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containing disinfectants showed partial amoebicidal effects and the number of viable amoeba
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was 7.35 x 104 ± 3.55 x 104, 4.95 x 104 ± 5.5 x 103 for Oxysept®, and AO SEPT PLUS
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respectively (Fig. 2).
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To determine whether the addition of cellulase and chlorhexidine at micromolar
189
concentration improve the efficacy of contact lens disinfectants, cellulase enzyme was added
190
at 70 µM. The results revealed that the addition of cellulase showed no effect on the viability
191
of trophozoites and after six hours of incubation the number of viable amoebae was 3.28 x
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105 ± 3.5 x 103. When cellulase was combined with contact lens disinfectants, OPTI-FREE®
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pure moist®, Renu® freshTM, FreshKon® CLEAR and COMPLETE RevitaLensTM the number
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of viable amoeba was 2 x 105 ± 2 x 103, 2.68 x 105 ± 4.0 x 104, 2.67 x 105 ± 1.1 x 104 and
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2.66 x 105 ± 1.5 x 104 respectively but upon re-inoculation in fresh PYG, viable trophozoites
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emerged within 72 h. Similarly, chlorhexidine alone (up to 50µM) showed potent
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amoebicidal effects on the viability of A. castellanii trophozoites. Notably, contact lens disinfectants, OPTI-FREE® pure moist®, Renu® freshTM ,
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FreshKon® CLEAR and COMPLETE RevitaLensTM in combination with cellulase (70 units)
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and chlorhexidine (50µM) abolished viability of A. castellanii trophozoites within 6 h of
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incubation and the number was reduced to 3.85 x 104 ± 1.2 x 104, 1.50 x 104 ± 4.00 x 103,
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3.10 x 104 ± 9 x 103 and 3 x 104 ± 1 x 103 respectively (Fig. 2). Post-treatment, amoebae were
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unable to revive in fresh PYG medium even after 72 h (data not shown). Amoebae in RPMI
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as negative control and in methanol as solvent control for chlorhexidine showed no
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amoebicidal effect and the number was 5.52 x 105 ± 6.5 x 104 and 4.01 x 105 ± 5.3 x 104
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respectively.
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Addition of cellulase and chlorhexidine improved efficacy of contact lens disinfectants
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against cysts of A. castellanii
Cysticidal assays were performed to evaluate the efficacy of contact lens
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disinfectants. The results revealed that all four multipurpose Contact lens disinfectant
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(without hydrogen peroxide) OPTI-FREE® pure moist®, Renu® freshTM, FreshKon® CLEAR
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and COMPLETE RevitaLensTM were ineffective in killing A. castellanii cyst (Fig. 3), and
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viable amoebae emerged when inoculated in fresh PYG compared with the original inoculum
215
(dotted line). Post-treatment, viable trophozoites emerged within 72 h. Hydrogen peroxide
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containing disinfectants (Oxysept® and AO SEPT PLUS) showed partial cysticidal effects
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(Fig. 3). Cellulase enzyme when used at 70µM showed no significant effect and the number 9
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was 1.72 x 105 ± 1.69 x 104. However, combination of cellulase and chlorhexidine with
219
contact lens disinfectants abolished viability of A. castellanii cysts and amoebae were unable
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to revive when inoculated in fresh PYG (Fig. 3).
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Discussion
Keratitis is a vision-threatening disease of the eye often associated with the use of contact lens (Mascarenhas et al., 2015). If not treated promptly, it can lead to permanent
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vision impairment. Improper contact lens hygiene, use of homemade saline solutions for
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cleaning and storing lenses as well as using expired disinfectants further leads to an increase
227
risk of microbial keratitis among contact lens wearers (Stapleton et al., 2007; 2012; Lorenzo-
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Morales et al., 2013). In this study, we determine the effectiveness of contact lens
229
disinfectants available commercially in Malaysia against common bacterial eye pathogens
230
and corneal parasite, Acanthamoeba castellanii. Our finding revealed that all contact lens
231
disinfectants tested exhibited potent bactericidal effects against species tested. However, none
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of the contact lens disinfectants completely destroyed A. castellanii trophozoites and cyst.
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These findings are consistent with several recent studies indicating that marketed contact lens
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disinfectants are ineffective in completely destroying amoebae (Kolar et al., 2015; Padzik et
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al., 2014; Polat et al., 2007; Ustüntürk and Zeybek, 2014). In particular, there show limited
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efficacy against cysts. Chlorhexidine at 50 µM concentration destroyed amoeba within 6 h of
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incubation time at room temperature. However, cellulase treatment had no effect on the
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viability of amoebae and parasites re-emerged as viable trophozoites upon re-inoculation in
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PYG. In contrast, a combination of chlorhexidine and cellulase together with contact lens
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disinfectants proved highly effective in killing both A. castellanii cyst and trophozoites. Our
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findings showed that targeting the cellulose synthesis pathway inhibits Acanthamoeba
242
switching into the cyst form. This is likely due to the ability of cellulase to destroy cyst walls
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ultimately leading to its death. This is consistent with our previous findings which showed
245
that the addition of cellulase enzyme together with chlorhexidine enhanced the efficacy of
246
contact lens solutions marketed in Pakistan (Abjani et al., 2016). As cellulose synthesis is
247
limited to plants, yeast, and some bacteria, it is likely to have minimal side effects against
248
human cells. Overall, the results obtained from our previous as well as present study clearly
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demonstrate that there is a need for improved contact lens disinfectant against Acanthamoeba
250
castellanii. As cyst are partly made of cellulose, degradation of cellulose by cellulase enzyme
251
and chlorhexidine is a promising strategy in targeting A. castellanii trophozoites as well as
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cysts. Our findings also highlight the ineffectiveness of commercially available contact lens
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disinfectant in Malaysia that present a major health risk to the public. We propose that similar
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strategy can be employed in the improved treatment of Acanthamoeba keratitis that can
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normally last up to a year and often complicated due to infection recurrence (Clarke et al.,
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2012), however intensive research is needed over the next few years to determine the
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translational value of these findings.
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Conflicts of interest: None to declare.
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Acknowledgments: The authors are grateful for the kind support provided by Sunway
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University, Malaysia and Lancaster University, UK.
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ACCEPTED MANUSCRIPT References
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Abjani, F., Khan, N.A., Yousuf, F.A. and Siddiqui, R., 2016. Targeting cyst wall is an
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effective strategy in improving the efficacy of marketed contact lens disinfecting
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solutions against Acanthamoeba castellanii cysts. Cont Lens Anter Eye, 39(3), 239-
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Aksozek, A., McClellan, K., Howard, K., Niederkorn, J.Y., Alizadeh, H., 2002. Resistance of Acanthamoeba castellanii cysts to physical, chemical, and radiological conditions. J
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Ansari, Z., Miller, D., Galor, A., 2013. Current thoughts in fungal keratitis: diagnosis and treatment. Curr Fungal Infect Rep 7, 209-218.
Clarke, B., Sinha, A., Parmar, D.N., Sykakis, E., 2012. Advances in the diagnosis and treatment of Acanthamoeba keratitis. J Ophthalmol Article ID: 484892.
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Lakhundi, S., Khan, N.A. and Siddiqui, R., 2014. Inefficacy of marketed contact lens
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Lorenzo-Morales, J., Martín-Navarro, C.M., López-Arencibia, A., Arnalich-Montiel, F.,
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Piñero, J.E., Valladares, B., 2013. Acanthamoeba keratitis: an emerging disease
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Marciano-Cabral, F., Cabral G., 2003. Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 16(2), 273-307. Mascarenhas, J., Lalitha, P., Prajna, N.V., Srinivasan, M., Das, M., D'Silva, S.S., Oldenburg,
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C.E., Borkar, D.S., Esterberg, E.J., Lietman, T.M., Keenan, J.D., 2014. Acanthamoeba,
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fungal, and bacterial keratitis: a comparison of risk factors and clinical features. Am J
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Ophthalmol 157(1), 56-62.
Padzik, M., Chomicz, L., Szaflik, J.P., Chruścikowska, A., Perkowski, K., Szaflik, J., 2014.
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In vitro effects of selected contact lens care solutions on Acanthamoeba castellanii
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strains in Poland. Exp Parasitol 145 Suppl., S98-S101.
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Polat, Z.A., Vural, A., Cetin, A., 2007. Efficacy of contact lens storage solutions against
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trophozoite and cyst of Acanthamoeba castellanii strain 1BU and their cytotoxic
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Sissons, J., Kim, K.S., Stins, M., Jayasekera, S., Alsam, S., Khan, N.A., 2005. Acanthamoeba
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Relationship between climate, disease severity, and causative organism for contact
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lens-associated microbial keratitis in Australia. Am J Ophthalmol 144, 690-698.
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Stapleton, F., Carnt, N., 2012. Contact lens-related microbial keratitis: how have
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epidemiology and genetics helped us with pathogenesis and prophylaxis. Eye (Lond)
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26, 185-193.
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Turner, N.A., Russell, A.D., Furr, J.R., Lloyd, D., 2004. Resistance, biguanide sorption and
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biguanide-induced pentose leakage during encystment of Acanthamoeba castellanii. J
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Appl Microbiol 96(6), 1287-1295.
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Ustüntürk, M., Zeybek, Z., 2014. Amoebicidal efficacy of a novel multi-purpose disinfecting solution: first findings. Exp Parasitol 145 Suppl., S93-97.
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Visvesvara, G.S., Moura, H., Schuster, F.L., 2007. Pathogenic and opportunistic free-living
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amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and
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Sappinia diploidea. FEMS Immunol Med Microbiol 50, 1-26.
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Figure Legends
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Figure 1. Commonly used contact lens disinfectants marketed in Malaysia were tested
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including Oxysept (OS), AO SEPT PLUS (AO SP), OPTI-FREE pure moist (OF PM), Renu
324
fresh (RF), FreshKon CLEAR (FKC) and COMPLETE RevitaLens (CRL) against MRSA
325
(A), P. aeruginosa (B), Streptococcus pyogenes (C), and Streptococcus pneumoniae (D)
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using Manufacturer’s Minimum Recommended Disinfection time (MRDT) as described in
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Materials and Methods. 100 µg/ml of gentamicin was used as a positive control, while
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bacteria incubated in PBS alone were used as a negative control. All six disinfectants tested
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abolished bacterial viability. The data is representative of several experiments performed in
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duplicate as expressed as the mean ± standard error.
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Figure 2. Contact lens disinfectants including Oxysept (OS), AO SEPT PLUS (AO SP),
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OPTI-FREE pure moist (OF PM), Renu fresh (RF), FreshKon CLEAR (FKC) and
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COMPLETE RevitaLens (CRL) were tested against A. castellanii trophozoites as described
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in Materials and Methods. Note that none of the contact lens disinfectants destroyed amoebae
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effectively. However, addition of chlorhexidine (CHX) and cellulase to contact lens
337
disinfectant abolished A. castellanii viability as compared to the contact lens disinfectant
338
alone. The dotted line represents initial inoculum of trophozoites. The data is expressed as the
339
mean ± standard error of several experiments performed in duplicate.
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Figure 3. Contact lens disinfectants including Oxysept (OS), AO SEPT PLUS (AO SP),
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OPTI-FREE pure moist (OF PM), Renu fresh (RF), FreshKon CLEAR (FKC) and
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COMPLETE RevitaLens (CRL) were tested against A. castellanii cysts as described in
344
Materials and Methods. Note that none of the contact lens disinfectants destroyed amoebae
345
cysts. However, addition of chlorhexidine (CHX) and cellulase to contact lens disinfectant
346
abolished A. castellanii viability as compared to the contact lens disinfectant alone. The
347
dotted line represents initial inoculum of cysts. The data is expressed as the mean ± standard
348
error of several experiments performed in duplicate.
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ACCEPTED MANUSCRIPT Table 1 List of contact lens disinfectants tested in the present study together with their ingredients, manufacturer, type of solution and Minimum recommended disinfection time (MRDT). Ingredients
Manufacturer
Oxysept®
3% Hydrogen peroxide
Abbot Medical Optics, Hangzhou
AO SEPT PLUS
3% Hydrogen peroxide
Alcon laboratories, Inc. Fort Worth, USA.
6h
Hydrogen peroxide system
OPTI-FREE® PureMoist®
POLYQUAD® (polyquaternium-1) 0.001% and ALDOX® (Myristamidopropyl dimethylamine) 0.0006% preservatives
Alcon laboratories, Inc. Fort Worth, Texas USA.
6h
Multi-purpose Disinfecting solution
COMPLETE RevitaLensTM
Alexidine dihydrochloride 0.00016%, Polyquaternium-1 0.0003%, boric acid, sodium borate decahydrate, TETRONIC 904, edetate disodium, sodium citrate, sodium chloride and purified water.
Abbot Medical Optics, Hangzhou
6h
Multi-purpose Disinfecting solution
Renu® freshTM
HYDRANATE Baush & Lomb (hydroxyalkylphosphonate), boric Greenville, acid, edetate disodium, poloxamine, USA. sodium borate and sodiumchloride, preserved with DYMEDTM (polyaminopropyl biguanide) 0.0001%
4h
Multi-purpose solution
Sodium and Potassium chloride, Disodium Edetate, Polyhexanide, Poloxamer, Hypromellose and Sodium Phosphate Buffer
6h
Multi-purpose solution
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FreshKon® CLEAR
Minimum Type of recommended solution disinfection time (MRDT) 6h Hydrogen peroxide system
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Solution
Oculus private limited, Singapore
ACCEPTED MANUSCRIPT
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Fig. 1A
1.80E+09
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1.60E+09
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1.20E+09 1.00E+09 8.00E+08
2.00E+08 0.00E+00 PBS
Gentamycin
MRSA
Renu fesh
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4.00E+08
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6.00E+08
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MRSA (CFU/ml)
1.40E+09
FreshKon CLEAR
OPTI-FREE pure moist
COMPLETE RevitaLens
MRSA + Contact Lens Disinfectant
Oxysept
AO SEPT PLUS
ACCEPTED MANUSCRIPT
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Fig. 1B
3.50E+09
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2.50E+09 2.00E+09 1.50E+09
5.00E+08 0.00E+00 PBS
Gentamycin
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1.00E+09
Renu fesh
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P. aeruginosa (CFU/ml)
3.00E+09
P. aeruginosa
FreshKon CLEAR
OPTI-FREE pure moist
COMPLETE RevitaLens
Oxysept
P. aeruginosa + Contact Lens Disinfectant
AO SEPT PLUS
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Fig. 1C
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1.40E+09
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1.00E+09 8.00E+08 6.00E+08
2.00E+08 0.00E+00 PBS
Gentamycin
S. pyogenes
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4.00E+08
Renu fesh
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S. pyogenes (CFU/ml)
1.20E+09
FreshKon CLEAR
OPTI-FREE pure moist
COMPLETE RevitaLens
S. pyogenes + Contact Lens Disinfectant
Oxysept
AO SEPT PLUS
ACCEPTED MANUSCRIPT
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Fig. 1D
3.00E+09
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2.00E+09 1.50E+09
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1.00E+09
0.00E+00 PBS
Gentamycin
Renu fesh
EP
5.00E+08
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S. Pneumoniae (CFU/ml)
2.50E+09
S. pneumoniae
FreshKon CLEAR
OPTI-FREE pure moist
COMPLETE RevitaLens
Oxysept
S. pneumoniae + Contact Lens Disinfectant
AO SEPT PLUS
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Fig. 2
P < 0.05
P < 0.05
5.00E+05 P < 0.05
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4.00E+05 3.00E+05
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0.00E+00
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2.00E+05 1.00E+05
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P < 0.05
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Number of viable Acanthamoeba
6.00E+05
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7.00E+05
ACCEPTED MANUSCRIPT
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Fig. 3
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2.50E+05
P < 0.05
P < 0.05 P < 0.05 P < 0.05
M AN U
2.00E+05
1.50E+05
0.00E+00
EP
5.00E+04
TE D
1.00E+05
AC C
Number of Acanthamoeba.castellanii
3.00E+05
ACCEPTED MANUSCRIPT
Highlights Efficacy of contact lens solutions was determined against keratitis-causing microbes.
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Of the six contact lens solutions tested, all were effective in killing common bacterial eye pathogens.
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None of them showed any cysticidal effects.
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Addition of cellulase and chlorhexidine improved efficacy of CL solutions
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