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Status of the effectiveness of contact lens solutions against keratitis-causing pathogens
Contact Lens & Anterior Eye 38 (2015) 34–38
Contents lists available at ScienceDirect
Contact Lens & Anterior Eye journal homepage: www.elsevier.com/locate/clae
Status of the effectiveness of contact lens solutions against keratitis-causing pathogens Ruqaiyyah Siddiqui, Sahreena Lakhundi, Naveed Ahmed Khan ∗ Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan
a r t i c l e
i n f o
Article history: Received 8 July 2014 Received in revised form 22 August 2014 Accepted 9 September 2014 Keywords: Microbial keratitis Contact lens Cleaning solution
a b s t r a c t Purpose: The aim of this study was to assess the antimicrobial effects of marketed contact lens disinfecting solutions. Methods: Using ISO 14729 Stand-Alone Test for disinfecting solutions, bactericidal, fungicidal and amoebicidal assays of eight different contact lens solutions including: ReNu MultiPlus, DuraPlus, Ultimate Plus, OptiFree Express, Kontex Clean, Kontex Normal, Kontex Multisol extra+ , Kontex Soak were performed. The efficacy of contact lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Fusarium solani and Acanthamoeba castellanii. Results: The results revealed that ReNu MultiPlus, DuraPlus and OptiFree Express were effective in killing bacterial and fungal pathogens as per manufacturer’s minimum recommended disinfection time. Ultimate Plus was effective against F. solani and MRSA but ineffective against P. aeruginosa, S. marcescens and S. aureus. Of concern however, is that none of the locally formulated contact lens disinfecting solutions from Pakistan, i.e., Kontex Clean, Kontex Normal, Kontex Multisol extra+ and Kontex Soak were effective against any of the keratitis-causing organisms tested. All eight contact lens disinfecting solutions were unable to destroy Acanthamoeba cysts. Conclusions: Because such ineffective contact lens disinfection solutions present a major risk to public health, these findings are of great concern to the health officials and to the manufacturers of the contact lens disinfection solutions and effective solutions are needed, along with emphasis on proper hygiene for contact lens care and special guidelines for developing countries regarding the manufacture and storage of contact lens disinfecting solutions. © 2014 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.
1. Introduction Microbial keratitis is a devastating ocular infection and an important cause of visual impairment/blindness that is frequently associated with contact lens (CL) wear [1–4]. The majority of cases are attributed to Pseudomonas aeruginosa, Serratia, Fusarium spp., and Acanthamoeba spp. [1–4]. The recognition and management of microbial keratitis require suspicion, early differential diagnosis and aggressive treatment for successful prognosis otherwise it often has vision-threatening consequences. With over 120 million people wearing CL for refractive correction and cosmetic purposes throughout the world, the associated
∗ Corresponding author at: Department of Biological and Biomedical Sciences, Aga Khan University, Stadium Road, Karachi, Pakistan. Tel.: +92 021 3486 4540; fax: +92 021 3493 4294. E-mail address: [email protected] (N.A. Khan).
risk factors are a cause for concern. Recently the anti-amoebic effects of marketed CL disinfecting solutions were assessed [5]. The efficacies of different CL disinfecting solutions manufactured locally in Pakistan and internationally were evaluated against Acanthamoeba castellanii of the T4 genotype. Surprisingly, none of the solutions tested had any potent cysticidal effects [5]. Following this alarming finding, the aim of the present study was to determine the antibacterial and antifungal efficacy of different CL disinfecting solutions against a range of bacterial and fungal pathogens: P. aeruginosa, Serratia marcescens, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus and Fusarium solani. Using the ISO 14729 Stand-Alone Test for CL disinfecting solutions, bactericidal and fungicidal effects of eight different CL disinfecting solutions including: ReNu MultiPlus, DuraPlus, Ultimate Plus, OptiFree Express, Kontex Clean, Kontex Normal, Kontex Multisol extra+ , Kontex Soak was performed. To allow comparison to our previous data of Acanthamoeba cysts, the CL disinfecting solutions were tested against A. castellanii of the T4 genotype.
http://dx.doi.org/10.1016/j.clae.2014.09.001 1367-0484/© 2014 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.
R. Siddiqui et al. / Contact Lens & Anterior Eye 38 (2015) 34–38
2. Materials and methods 2.1. Contact lens disinfecting solutions The CL disinfection solutions used in this study along with their active ingredients and manufacturers’ instructions are listed in Table 1. All CL disinfection solutions were tested within their stated expiry date. Eight different CL disinfection solutions were tested: ReNu MultiPlus, DuraPlus, Ultimate Plus, OptiFree Express, Kontex Clean, Kontex Normal, Kontex Multisol extra+ , Kontex Soak. These solutions are commonly available in Pakistan and were purchased from local retailers. ReNu MultiPlus and OptiFree Express were imported from the USA, whereas both DuraPlus and Ultimate Plus were manufactured locally, though were from international manufacturers’. Kontex Clean, Kontex Normal, Kontex Multisol extra+ , and Kontex Soak were formulated and manufactured in Karachi, Pakistan. 2.2. Test organisms and growth conditions All chemicals were purchased from Sigma Labs (Poole, Dorset, England), unless otherwise stated. P. aeruginosa, S. marcescens, S. aureus, MRSA and F. solani were used in this study. P. aeruginosa was isolated from a clinical sample at the Aga Khan University hospital. S. marcescens was isolated from the gut of Black Cobra [6]. S. aureus was obtained from an environmental sample at the Aga Khan University [7]. MRSA was isolated from the blood sample of a sepsis patient [8]. F. solani was purchased from the First Fungal Culture Bank of Pakistan (FCBP0055). All bacterial strains were grown on nutrient agar at 37 ◦ C for 24 h. Cultures were prepared by inoculating each bacterial strain in Luria–Bertani (LB) broth overnight and incubating at 37 ◦ C. Cultures were centrifuged 10,000 × g for 10 min and resuspended in phosphate-buffered saline (PBS), and adjusted to optical density of 0.2 spectrophotometrically, which is equivalent to approximately 108 colony forming units (CFU)/mL as well as enumerated by plating on nutrient agar plates using serial dilution [9,10]. F. solani was grown for 7–10 days on potato dextrose agar at 30 ◦ C [11]. Following incubation, conidia were harvested by scraping the surface of fungal colony with a cell scraper and hyphae were removed via filtration using sterile gauze [9–11]. The conidia were washed with sterile PBS and counted using a haemocytometer. A. castellanii belonging to the T4 genotype (ATC 50492) sourced from a keratitis patient was grown in PYG medium [proteose peptone 0.75% (w/v), yeast extract 0.75% (w/v) and glucose 1.5% (w/v)] at 30 ◦ C as previously described [12]. To prepare A. castellanii cysts, encystation was induced by inoculating 5 × 106 A. castellanii trophozoites onto non-nutrient agar plates and incubating at 30 ◦ C for up to 14 days [13]. Food deprivation resulted in trophozoite transformation into the cyst form. Next, 10 mL of dH2 O was added to each plate. Cysts were then scraped off the agar surface using a cell scraper, enumerated using a haemocytometer and 5 × 104 A. castellanii cysts were used. 2.3. Bactericidal activity of contact lens disinfection solutions To determine the effects of various CL solutions against P. aeruginosa, S. marcescens, S. aureus and MRSA, bactericidal assays were performed. The assay method was based on ISO 14729 StandAlone Test criteria for testing CL disinfecting solutions [9,10]. Briefly, 106 CFU/mL of test organisms were exposed to 10 mL of different CL disinfection solutions and incubated at room temperature as per manufacturer’s minimum recommended disinfection time. Following Kontex products were used: Kontex Clean is used to disinfect; Kontex Normal is used for rinsing; Kontex Multisol extra+ is used for extra rinsing; and Kontex Soak is used for soaking/storage. The tested organisms were first suspended in Kontex Clean for
35
30 s followed by centrifugation at 10,000 × g for 5 min. The supernatants were aspirated and organisms rinsed in Kontex Normal and/or Kontex Multisol extra rinsing solutions as recommended by the manufacturer. Next, 10-fold serial dilutions were made using Dey–Engley neutralizing broth after the manufacturer’s minimum recommended disinfectant time. The bacterial (CFU) were determined by plating dilutions on nutrient agar plates and incubating at 37 ◦ C for 24 h. All experiments were performed at least 3 times, in duplicate. 2.4. Fungicidal and amoebicidal activity of contact lens disinfection solutions To study the effects of CL disinfection solutions against F. solani, fungicidal assays were performed. The assay method was based on ISO 14729 Stand-Alone Test criteria for testing CL disinfecting solutions [9,10]. Briefly, 106 CFU of fungal conidia were exposed to 10 mL of different CL disinfecting solutions and incubated at room temperature as per the manufacturer’s minimum recommended disinfection time. In the case of Kontex Multisol extra+ and Kontex Soak, conidia were first exposed to Kontex Clean solution for 30 s followed by centrifugation at 4500 × g for 5 min. The supernatants were aspirated and conidia rinsed in Kontex Normal and/or Kontex Multisol extra rinsing solutions as recommended by the manufacturer. Next, 10-fold serial dilutions were made using Dey–Engley neutralizing broth and mixtures allowed to stand at room temperature for 10–15 min to neutralize the preservatives. The conidia were enumerated by plating the dilutions on potato dextrose agar plates and incubating at 30 ◦ C for up to 5 days. All experiments were performed at least 3 times, in duplicate. Additionally, the cysticidal effectiveness of these solutions was determined against A. castellanii. Briefly, 5 × 104 A. castellanii cysts were pelleted by centrifugation at 2500 × g for 5 min. The supernatants were aspirated and pellet resuspended in 0.5 mL of each CL disinfection solution listed in Table 1. In the case of Kontex Multisol extra+ and Kontex Soak, amoebae were first suspended in Kontex Clean for 30 s followed by centrifugation at 2500 × g for 5 min. The supernatants were aspirated and amoebae resuspended in Kontex Normal and/or Kontex Multisol extra+ as recommended by the manufacturer. Next, amoebae counts were performed using a haemocytometer after the manufacturer’s minimum recommended disinfectant time as well as after 24 h. The viability of trophozoites was determined by inoculating CL disinfection solution-treated amoebae in the growth medium, i.e., PYG and incubating at 30 ◦ C for 72 h [5]. 3. Results 3.1. Bactericidal effects of contact lens disinfection solutions tested The results revealed that ReNu MultiPlus, DuraPlus and OptiFree Express killed P. aeruginosa, S. marcescens, S. aureus and MRSA when tested for the manufacturers’ recommended disinfection time. More than 3-log reduction was observed in case of these CL disinfection solutions against the tested bacteria (Fig. 1 and Table 2). On the other hand, Ultimate Plus was effective against MRSA with a 3-log reduction, but not against P. aeruginosa, S. marcescens and S. aureus. When tested against P. aeruginosa, S. marcescens and S. aureus, 2.7-, 2.6- and 1.7-log reductions were observed respectively (Fig. 1 and Table 2), which is considered ineffective according to ISO Stand-Alone Test primary acceptance criteria [9]. Notably, the locally formulated solutions: Kontex-CLS (i.e., Kontex Clean, Kontex Normal, Kontex Multisol extra+ and Kontex Soak) did not exhibit any bactericidal activity against any of the bacterial
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R. Siddiqui et al. / Contact Lens & Anterior Eye 38 (2015) 34–38
Table 1 Contact lens solutions tested in the present study together with their ingredients and manufacturers’ minimum recommended disinfection time. Solution
Type
Ingredients
Minimum recommended disinfection time
Manufacturer
ReNu MultiPlus
Multi-purpose solution
4h
Bausch & Lomb, Greenville, USA.
DuraPlus
Multi-purpose solution
HYDRANATE® (hydroxyalkylphosphonate), boric acid, edetate disodium, POLOXAMINE, sodium borate and sodium chloride; preserved with DYMEDTM (polyaminopropyl biguanide) 0.0001% Edetate Disodium, Poloxamine, Sodium Chloride and Aquahydrate TM ; preserved with OPTIMED (polyaminopropyl biguanide). Contains no chlorhexidine, no thimerosal and no sorbic acid.
4h
Ultimate Plus
Multi-purpose solution
Polyhexamethylene biguanide, tromethamine, tyloxapol, hydroxy propyl methylcellulose (HPMC) and edetate disodium.
4h
OptiFree Express
Multi-purpose disinfecting solution
6h
Kontex Clean + Kontex Multisol extra+
Soft contact lens daily cleaning solution for sensitive eyes Soft contact lens daily cleaning, soaking, storing and cold disinfecting solution for sensitive eyes
Sodium chloride, sorbitol, edetate disodium, boric acid, aminomethyl propanol, citrate and tetronic® (polidronium chloride) 0.001% and ALDOX® (myristamidopropyl dimethylamine) 0.0005% EDTA 0.2% soft surfact. 8 polymer WS 2% in preserved isotonic solution + EDTA 0.01% citrate, poloxamer WS, S. borate, Naci, SRFACT X, HPG 0.0005% (TN) preservative. EDTA 0.2% softsurfact. 8 polymer WS 2% in preserved isotonic solution + EDTA 0.01% citrate, poloxamer WS, S. borate, Naci, SRFACT X, HPG 0.0005% (TN) preservative + borsaure M61.83 g/mol extra pure DAB, thimerosal 0.001 mg/mL, ethylenediamine tetra-A sodium salt 1 mg/mL, HPG (high preservative grades). EDTA 0.2% softsurfact. 8 polymer WS 2% in preserved isotonic solution + Isotonic buffered saline solution + EDTA 0.01% citrate, poloxamer WS, S. borate, Naci, SRFACT X, HPG 0.0005% (TN) preservative. EDTA 0.2% softsurfact. 8 polymer WS 2% in preserved isotonic solution + isotonic buffered saline solution + borsaure M61.83 g/mol extra pure DAB, thimerosal 0.001 mg/mL, ethylenediamine tetra-A sodium salt 1 mg/mL, HPG (high preservative grades).
SIPIC INTERNATIONAL Texas, U.S.A (International formulation, manufactured locally) ELKO Organization (Pvt.) Ltd. Switzerland (International formulation, manufactured locally in Karachi, Pakistan). Alcon Laboratories, Inc. Fort Worth, USA.
Kontex Clean + Kontex Multisol extra+ + Kontex Soak
Kontex Clean + Kontex Normal + Kontex Multisol extra+ Kontex Clean + Kontex Normal + Kontex Soak
Soft contact lens daily cleaning, rinsing and storing saline solution for sensitive eyes Soft contact lens daily cleaning, rinsing, soaking, storing and cold disinfecting solution
pathogens tested (Fig. 1 and Table 2). When bacteria treated with Kontex-CLS were re-inoculated on to nutrient agar plates, viable bacteria emerged in all cases and no reduction in the number of viable organisms was observed (Fig. 1 and Table 2). For CL disinfection solutions: Kontex Clean + Kontex Normal + Kontex Soak, 0-, 0.4-, 0- and 0-log reduction was observed against of P. aeruginosa,
30 s + quick rinse + soaking for 4h 30 s + quick rinse + soaking for 4h
KONTEX Corp. Laboratories, Karachi, Pakistan. KONTEX Corp. Laboratories, Karachi, Pakistan.
30 sec + quick rinse + soaking for 4h
KONTEX Corp. Laboratories, Karachi, Pakistan.
30 s + quick rinse + soaking for 4h
KONTEX Corp. Laboratories, Karachi, Pakistan.
S. marcescens, S. aureus and MRSA respectively when tested for the manufacturers’ recommended disinfection time. The combination of Kontex Clean + Kontex Normal + Kontex Multisol Extra revealed 0-, 0.4-, 0.03- and 0-log reductions when tested against P. aeruginosa, S. marcescens, S. aureus and MRSA respectively. Similarly when the combination of Kontex Clean + Kontex Multisol
Fig. 1. The efficacy of contact lens solutions against Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus. Eight different types of contact lens solutions were tested to determine their effectiveness against different bacteria according to manufacturer’s minimum recommended disinfection time as described in Materials and Section 2. Phosphate buffered saline was used as a control. White bars indicate P. aeruginosa, light grey bars indicate S. marcescens, dark grey bars indicate S. aureus and black bars indicate MRSA, treated with different contact lens solutions. The results are representative of three individual experiments performed in duplicate. The data are presented as the mean ± standard error.
R. Siddiqui et al. / Contact Lens & Anterior Eye 38 (2015) 34–38
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Table 2 The mean-log reduction of microbes tested, post-exposure of contact lens disinfectant solution as per manufacturers’ minimum recommended disinfection time. Contact lens solutions
ReNu MultiPlus DuraPlus Ultimate Plus OptiFree Express Kontex Clean + Kontex Normal + Kontex Soak Kontex Clean + Kontex Normal + Kontex Multisol Extra Kontex Clean + Kontex Multisol Extra + Kontex Soak Kontex Clean + Kontex Multisol Extra
Log reduction (standard deviation) P. aeruginosa
S. marcescens
S. aureus
MRSA
F. solani
5.13 (0.103) 5.13 (0.115) 2.72 (0.08) 5.13 (0.23) −0.24 (0.01) −0.36 (0.02) −0.43 (0.006) −0.36 (0.07)
5.76 (0.112) 5.76 (0.15) 2.61 (0.1) 5.76 (0.151) 0.38 (0.1) 0.35 (0.01) 0.20 (0.1) 0.27 (0.03)
4.41 (0.1) 4.41 (0.117) 1.74 (0.04) 4.41 (0.183) −0.07 (0.008) 0.03 (0.04) 0.02 (0.01) −0.05 (0)
4.56 (0.101) 4.56 (0.1) 4.56 (0.153) 4.56 (0.114) −0.13 (0.03) −0.04 (0.004) −0.12 (0.005) −0.15 (0.004)
5.79 (0.165) 5.79 (0.101) 2.71 (0.08) 5.79 (0.187) 0.34 (0.07) 0.32 (0.1) −0.10 (0.02) −0.05 (0.02)
Fig. 2. The efficacy of contact lens solutions against Fusarium solani. F. solani (106 conidia) were incubated with different contact lens solutions at room temperature for manufacturer’s minimum recommended disinfection time. Following this, conidia were plated onto the potato dextrose agar plates. The results represent the mean ± standard error of three independent experiments performed in duplicate.
Extra + Kontex Soak was used, 0-, 0.2-, 0.02- and 0-log reduction was observed against P. aeruginosa, S. marcescens, S. aureus and MRSA. The combination of Kontex Clean + Kontex Multisol Extra showed 0-, 0.3-, 0- and 0-log reductions when tested against P. aeruginosa, S. marcescens, S. aureus and MRSA respectively (Fig. 1 and Table 2). 3.2. Fungicidal and amoebicidal properties of contact lens disinfection solutions against F. solani
criteria [9]. However other CL disinfection solutions: Kontex Clean, Kontex Normal, Kontex Multisol extra+ and Kontex Soak were completely ineffective against fungal spores and no fungicidal effects were observed (Fig. 2 and Table 2). As expected, none of the CL disinfection solutions tested destroyed A. castellanii cysts as per manufacturers’ instructions (Fig. 3). Upon inoculation in the growth medium, cysts re-emerged as viable trophozoites (data not shown). 4. Discussion
To determine the fungicidal effects of CL disinfection solutions on F. solani, assays were performed by inoculating 106 conidiospores in 10 mL of CL disinfection solution. Of the eight CL disinfection solutions tested, the results revealed that ReNu MultiPlus, DuraPlus, Ultimate Plus and OptiFree Express were able to exhibit more than 1-log reduction in the number of viable fungal spores (Fig. 2 and Table 2). The cidal properties of ReNu MultiPlus, DuraPlus and OptiFree Express were most effective as no viable fungal colonies were seen on PDA, even after 5 days of incubation. In the case of Ultimate Plus, a 1.6-log reduction was observed which is effective according to ISO Stand-Alone primary acceptance
Contact lens wear is the most common risk factor in the development of microbial keratitis [14]. Studies to evaluate the efficacy of various types of CL disinfection solutions have been performed worldwide [15–19], whereas limited studies have been carried out for developing countries. This is particularly important as contact lens solutions are imported as well as formulated locally, and exposed to variable temperatures during storage or shipment and must be tested for their effectiveness. An increase in the incidence of microbial keratitis associated with the contact lens use at the Aga Khan University Hospital led us to hypothesize
Fig. 3. The efficacy of contact lens solutions against A. castellanii. Briefly, A. castellanii cysts (5 × 104 ) were incubated with different contact lens solutions at room temperature for manufacturer’s minimum recommended disinfection time. For controls, cysts were treated with PBS (negative control) and 1% SDS (amoebicidal effects). Next, cysts were enumerated using a haemocytometer as well as plated using growth medium (PYG). Note that none of the solutions tested had any effect on A. castellanii cysts. The results represent the mean ± standard error of three independent experiments performed in duplicate.
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R. Siddiqui et al. / Contact Lens & Anterior Eye 38 (2015) 34–38
ineffectiveness of the disinfecting solutions. Recently, the antiamoebic effects of commonly marketed CL disinfection solutions in Pakistan were tested. Astonishingly, none of the CL disinfection solutions tested had any cysticidal effects against A. castellanii keratitis isolate of the T4 genotype. Here, the efficacy of commonly marketed CL disinfection solutions against a range of bacterial and fungal pathogens was investigated. Of the eight tested solutions, ReNu MultiPlus, DuraPlus and OptiFree Express were found to be effective against all tested organisms including bacteria and fungi. On the other hand, Ultimate Plus failed to bring more than 3-log reduction in case of bacteria, including P. aeruginosa, S. marcescens and S. aureus. To our surprise, none of the locally formulated CL disinfection solutions: Kontex-CLS (Kontex Clean, Kontex Normal, Kontex Multisol extra+ and Kontex Soak) had any bactericidal and/or fungicidal effect against the corneal pathogens tested, which is a major concern for CL wearers. Additionally, none of the CL disinfection solutions were able to destroy A. castellanii cysts, which is consistent with previous studies [5]. The lack of efficacy of commonly marketed CL disinfection solutions in developing countries like Pakistan is a serious concern and suggests prevalence of microbial keratitis. As the locally formulated CL disinfection solutions are considerably cheaper to purchase, these findings may explain an increase in the number of cases of microbial keratitis (personal communication with the consultant ophthalmologists at the Aga Khan University Hospital, Karachi) suggesting the need for increased awareness. However, the actual burden of microbial keratitis remains unknown and requires further investigation. Notably, some of the known brands appeared ineffective in eradicating corneal pathogens. Previous studies have shown that the efficacy of CL disinfection solutions is affected by time and temperature of storage [20]. This may explain ineffectiveness of CL disinfection solutions tested in the present study, in addition possibly due to, lack of quality control during the manufacturing process or during importing, shipping and transportation of CL disinfection solutions and exposure to high temperatures. Manufacturers should consider this to safeguard the ocular health of contact lens wearers. Such ineffective CL disinfection solutions pose a serious risk to contact lens wearers and to the wider community. Additionally, of concern is the fact that these solutions can be purchased without any instructions given to the consumer, majority of who are unaware of the associated risks or they are teenagers in the case of cosmetic CL users. Moreover CL for cosmetic as well as corrective use are routinely available over the counter in developing countries such as Pakistan without any instructions of proper hygiene to handle CL given to the users. A study conducted at Aga Khan University, Karachi, reported that CL related corneal ulcers are resulting in serious visual impairment especially in women and many of the devastating outcomes of corneal infections are accredited to the use of contact lenses [21]. Overall, ineffective CL disinfection solutions present a major risk to public health; these findings are of great concern for health officials and to the manufacturers of the CL disinfection solutions. Travellers to developing countries who are CL wearers need to be aware of the associated risks when using any locally manufactured CL solutions. As well as being aware that the efficacy of solutions is affected by temperature. It is imperative that urgent intervention measures are implemented to address this issue.
Conflicts of interest None to declare. Acknowledgment The authors are grateful for the kind support provided by the Aga Khan University, Pakistan. References [1] Lam DSC, Houang E, Fan DSP, Lyon D, Seal D, Wong E, et al. Hong Kong Microbial Keratitis Study Group. Incidence and risk factors for microbial keratitis in Hong Kong: comparison with Europe and North America. Eye 2002;16:608–18. [2] Cheng KH, Leung SL, Hoekman HW, Beekhuis WH, Mulder PGH, Geerards AJM, et al. Incidence of contact-lens-associated microbial keratitis and its related morbidity. Lancet 1999;354(9174):181–5. [3] Poggio EC, Glynn RJ, Schein OD, Seddon JM, Shannon MJ, Scardino VA, et al. The incidence of ulcerative keratitis among users of daily-wear and extended-wear soft contact lenses. N Engl J Med 1989;321:779–83. [4] Mukherjee PK, Chandra J, Yu C, Sun Y, Pearlman E, Ghannoum MA. Characterization of Fusarium keratitis outbreak isolates: contribution of biofilms to antimicrobial resistance and pathogenesis. Immunol Microbiol 2012;55:4450–7. [5] Lakhundi S, Khan NA, Siddiqui R. Inefficacy of marketed contact lens disinfection solutions against keratitis-causing Acanthamoeba castellanii belonging to the T4 genotype. Exp Parasitol 2014;141:122–8. [6] Iqbal J, Sagheer M, Ahmed N, Siddiqui R, Khan NA. Culturable aerobic and facultative anaerobic intestinal bacterial flora of Black Cobra (Naja sp.). ISRN Vet Sci 2014 [article ID 878479] http://www.hindawi.com/journals/isrn/ 2014/878479/ [7] Siddiqui R, Sagheer M, Khan NA. Prevalence of Acanthamoeba and superbugs in a clinical setting: coincidence or hyperparasitism. Parasitol Res 2013;112:1349–51. [8] Cardas M, Khan NA, Alsam S. Staphylococcus aureus exhibit similarities in their interactions with Acanthamoeba and ThP1 macrophage-like cells. Exp Parasitol 2012;132:513–8. [9] International Standards Organization. ISO 14729 Ophthalmic optics – contact lens care products – microbiological requirements and test methods for products and regimens for hygienic management of contact lenses. Geneva, Switzerland: ISO; 2001. [10] Rosenthal RA, Sutton SW, Schlech BA. Review of standard for evaluating the effectiveness of contact lens disinfectants. PDAJ Pharm Sci Technol 2002;56:37–50. [11] Retuerto MA, Szczotka-Flynn L, Ho D, Mukherjee P, Ghannoum MA. Efficacy of care solutions against contact lens-associated Fusarium biofilms. Optom Vis Sci 2012;89:382–91. [12] Aqeel Y, Siddiqui R, Khan NA. Anti-Acanthamoebic properties of resveratrol and de-methoxy curcumin. Exp Parasitol 2012;132:519–23. [13] Dudley R, Jarroll EL, Khan NA. Carbohydrate analysis of Acanthamoeba castellanii. Exp Parasitol 2009;122:338–43. [14] Patel A, Hammersmith K. Contact lens-related microbial keratitis: recent outbreaks. Curr Opin Opthalmol 2008;19:302–6. [15] Borazjani RN, Kilvington S. Efficacy of multipurpose solutions against Acanthamoeba species. Contact Lens Anter Eye 2005;28:169–75. [16] Centers for Disease Control and Prevention. Acanthamoeba keratitis multiple states 2005–2007. MMWR: Morb Mortal Wkly Rep 2007;56:1–3. [17] Hughes R, Kilvington S. Comparision of hydrogen peroxide contact lens disinfection systems and solutions against Acanthamoeba polyphaga. Antimicrob Agents Chemother 2001;45:2038–43. [18] Miller MJ, Callahan DE, McGrath D, Manchester R, Norton SE. Disinfection efficacy of contact lens care solutions against ocular pathogens. CLAO J 2001;27:16–22. [19] Kobayashi T, Gibbon L, Mito T, Shiraishi A, Uno T, Ohashi Y. Efficacy of commercial soft contact lens disinfectant solutions against Acanthamoeba. Jpn J Ophthalmol 2011;55:547–57. [20] Leung P, Boost MV, Cho P. Effect of storage temperatures and time on the efficacy of multipurpose solutions for contact lenses. Ophthalmic Physiol Opt 2004;24:218–24. [21] Chaudhry TA, Sarfraz S, Khan QA, Ahmad K. Contact lens-related visual loss—a case series from Karachi. Pak Med Assoc 2011;61:1246–8.
Contents lists available at ScienceDirect
Contact Lens & Anterior Eye journal homepage: www.elsevier.com/locate/clae
Status of the effectiveness of contact lens solutions against keratitis-causing pathogens Ruqaiyyah Siddiqui, Sahreena Lakhundi, Naveed Ahmed Khan ∗ Department of Biological and Biomedical Sciences, Aga Khan University, Karachi, Pakistan
a r t i c l e
i n f o
Article history: Received 8 July 2014 Received in revised form 22 August 2014 Accepted 9 September 2014 Keywords: Microbial keratitis Contact lens Cleaning solution
a b s t r a c t Purpose: The aim of this study was to assess the antimicrobial effects of marketed contact lens disinfecting solutions. Methods: Using ISO 14729 Stand-Alone Test for disinfecting solutions, bactericidal, fungicidal and amoebicidal assays of eight different contact lens solutions including: ReNu MultiPlus, DuraPlus, Ultimate Plus, OptiFree Express, Kontex Clean, Kontex Normal, Kontex Multisol extra+ , Kontex Soak were performed. The efficacy of contact lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Fusarium solani and Acanthamoeba castellanii. Results: The results revealed that ReNu MultiPlus, DuraPlus and OptiFree Express were effective in killing bacterial and fungal pathogens as per manufacturer’s minimum recommended disinfection time. Ultimate Plus was effective against F. solani and MRSA but ineffective against P. aeruginosa, S. marcescens and S. aureus. Of concern however, is that none of the locally formulated contact lens disinfecting solutions from Pakistan, i.e., Kontex Clean, Kontex Normal, Kontex Multisol extra+ and Kontex Soak were effective against any of the keratitis-causing organisms tested. All eight contact lens disinfecting solutions were unable to destroy Acanthamoeba cysts. Conclusions: Because such ineffective contact lens disinfection solutions present a major risk to public health, these findings are of great concern to the health officials and to the manufacturers of the contact lens disinfection solutions and effective solutions are needed, along with emphasis on proper hygiene for contact lens care and special guidelines for developing countries regarding the manufacture and storage of contact lens disinfecting solutions. © 2014 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.
1. Introduction Microbial keratitis is a devastating ocular infection and an important cause of visual impairment/blindness that is frequently associated with contact lens (CL) wear [1–4]. The majority of cases are attributed to Pseudomonas aeruginosa, Serratia, Fusarium spp., and Acanthamoeba spp. [1–4]. The recognition and management of microbial keratitis require suspicion, early differential diagnosis and aggressive treatment for successful prognosis otherwise it often has vision-threatening consequences. With over 120 million people wearing CL for refractive correction and cosmetic purposes throughout the world, the associated
∗ Corresponding author at: Department of Biological and Biomedical Sciences, Aga Khan University, Stadium Road, Karachi, Pakistan. Tel.: +92 021 3486 4540; fax: +92 021 3493 4294. E-mail address: [email protected] (N.A. Khan).
risk factors are a cause for concern. Recently the anti-amoebic effects of marketed CL disinfecting solutions were assessed [5]. The efficacies of different CL disinfecting solutions manufactured locally in Pakistan and internationally were evaluated against Acanthamoeba castellanii of the T4 genotype. Surprisingly, none of the solutions tested had any potent cysticidal effects [5]. Following this alarming finding, the aim of the present study was to determine the antibacterial and antifungal efficacy of different CL disinfecting solutions against a range of bacterial and fungal pathogens: P. aeruginosa, Serratia marcescens, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus and Fusarium solani. Using the ISO 14729 Stand-Alone Test for CL disinfecting solutions, bactericidal and fungicidal effects of eight different CL disinfecting solutions including: ReNu MultiPlus, DuraPlus, Ultimate Plus, OptiFree Express, Kontex Clean, Kontex Normal, Kontex Multisol extra+ , Kontex Soak was performed. To allow comparison to our previous data of Acanthamoeba cysts, the CL disinfecting solutions were tested against A. castellanii of the T4 genotype.
http://dx.doi.org/10.1016/j.clae.2014.09.001 1367-0484/© 2014 British Contact Lens Association. Published by Elsevier Ltd. All rights reserved.
R. Siddiqui et al. / Contact Lens & Anterior Eye 38 (2015) 34–38
2. Materials and methods 2.1. Contact lens disinfecting solutions The CL disinfection solutions used in this study along with their active ingredients and manufacturers’ instructions are listed in Table 1. All CL disinfection solutions were tested within their stated expiry date. Eight different CL disinfection solutions were tested: ReNu MultiPlus, DuraPlus, Ultimate Plus, OptiFree Express, Kontex Clean, Kontex Normal, Kontex Multisol extra+ , Kontex Soak. These solutions are commonly available in Pakistan and were purchased from local retailers. ReNu MultiPlus and OptiFree Express were imported from the USA, whereas both DuraPlus and Ultimate Plus were manufactured locally, though were from international manufacturers’. Kontex Clean, Kontex Normal, Kontex Multisol extra+ , and Kontex Soak were formulated and manufactured in Karachi, Pakistan. 2.2. Test organisms and growth conditions All chemicals were purchased from Sigma Labs (Poole, Dorset, England), unless otherwise stated. P. aeruginosa, S. marcescens, S. aureus, MRSA and F. solani were used in this study. P. aeruginosa was isolated from a clinical sample at the Aga Khan University hospital. S. marcescens was isolated from the gut of Black Cobra [6]. S. aureus was obtained from an environmental sample at the Aga Khan University [7]. MRSA was isolated from the blood sample of a sepsis patient [8]. F. solani was purchased from the First Fungal Culture Bank of Pakistan (FCBP0055). All bacterial strains were grown on nutrient agar at 37 ◦ C for 24 h. Cultures were prepared by inoculating each bacterial strain in Luria–Bertani (LB) broth overnight and incubating at 37 ◦ C. Cultures were centrifuged 10,000 × g for 10 min and resuspended in phosphate-buffered saline (PBS), and adjusted to optical density of 0.2 spectrophotometrically, which is equivalent to approximately 108 colony forming units (CFU)/mL as well as enumerated by plating on nutrient agar plates using serial dilution [9,10]. F. solani was grown for 7–10 days on potato dextrose agar at 30 ◦ C [11]. Following incubation, conidia were harvested by scraping the surface of fungal colony with a cell scraper and hyphae were removed via filtration using sterile gauze [9–11]. The conidia were washed with sterile PBS and counted using a haemocytometer. A. castellanii belonging to the T4 genotype (ATC 50492) sourced from a keratitis patient was grown in PYG medium [proteose peptone 0.75% (w/v), yeast extract 0.75% (w/v) and glucose 1.5% (w/v)] at 30 ◦ C as previously described [12]. To prepare A. castellanii cysts, encystation was induced by inoculating 5 × 106 A. castellanii trophozoites onto non-nutrient agar plates and incubating at 30 ◦ C for up to 14 days [13]. Food deprivation resulted in trophozoite transformation into the cyst form. Next, 10 mL of dH2 O was added to each plate. Cysts were then scraped off the agar surface using a cell scraper, enumerated using a haemocytometer and 5 × 104 A. castellanii cysts were used. 2.3. Bactericidal activity of contact lens disinfection solutions To determine the effects of various CL solutions against P. aeruginosa, S. marcescens, S. aureus and MRSA, bactericidal assays were performed. The assay method was based on ISO 14729 StandAlone Test criteria for testing CL disinfecting solutions [9,10]. Briefly, 106 CFU/mL of test organisms were exposed to 10 mL of different CL disinfection solutions and incubated at room temperature as per manufacturer’s minimum recommended disinfection time. Following Kontex products were used: Kontex Clean is used to disinfect; Kontex Normal is used for rinsing; Kontex Multisol extra+ is used for extra rinsing; and Kontex Soak is used for soaking/storage. The tested organisms were first suspended in Kontex Clean for
35
30 s followed by centrifugation at 10,000 × g for 5 min. The supernatants were aspirated and organisms rinsed in Kontex Normal and/or Kontex Multisol extra rinsing solutions as recommended by the manufacturer. Next, 10-fold serial dilutions were made using Dey–Engley neutralizing broth after the manufacturer’s minimum recommended disinfectant time. The bacterial (CFU) were determined by plating dilutions on nutrient agar plates and incubating at 37 ◦ C for 24 h. All experiments were performed at least 3 times, in duplicate. 2.4. Fungicidal and amoebicidal activity of contact lens disinfection solutions To study the effects of CL disinfection solutions against F. solani, fungicidal assays were performed. The assay method was based on ISO 14729 Stand-Alone Test criteria for testing CL disinfecting solutions [9,10]. Briefly, 106 CFU of fungal conidia were exposed to 10 mL of different CL disinfecting solutions and incubated at room temperature as per the manufacturer’s minimum recommended disinfection time. In the case of Kontex Multisol extra+ and Kontex Soak, conidia were first exposed to Kontex Clean solution for 30 s followed by centrifugation at 4500 × g for 5 min. The supernatants were aspirated and conidia rinsed in Kontex Normal and/or Kontex Multisol extra rinsing solutions as recommended by the manufacturer. Next, 10-fold serial dilutions were made using Dey–Engley neutralizing broth and mixtures allowed to stand at room temperature for 10–15 min to neutralize the preservatives. The conidia were enumerated by plating the dilutions on potato dextrose agar plates and incubating at 30 ◦ C for up to 5 days. All experiments were performed at least 3 times, in duplicate. Additionally, the cysticidal effectiveness of these solutions was determined against A. castellanii. Briefly, 5 × 104 A. castellanii cysts were pelleted by centrifugation at 2500 × g for 5 min. The supernatants were aspirated and pellet resuspended in 0.5 mL of each CL disinfection solution listed in Table 1. In the case of Kontex Multisol extra+ and Kontex Soak, amoebae were first suspended in Kontex Clean for 30 s followed by centrifugation at 2500 × g for 5 min. The supernatants were aspirated and amoebae resuspended in Kontex Normal and/or Kontex Multisol extra+ as recommended by the manufacturer. Next, amoebae counts were performed using a haemocytometer after the manufacturer’s minimum recommended disinfectant time as well as after 24 h. The viability of trophozoites was determined by inoculating CL disinfection solution-treated amoebae in the growth medium, i.e., PYG and incubating at 30 ◦ C for 72 h [5]. 3. Results 3.1. Bactericidal effects of contact lens disinfection solutions tested The results revealed that ReNu MultiPlus, DuraPlus and OptiFree Express killed P. aeruginosa, S. marcescens, S. aureus and MRSA when tested for the manufacturers’ recommended disinfection time. More than 3-log reduction was observed in case of these CL disinfection solutions against the tested bacteria (Fig. 1 and Table 2). On the other hand, Ultimate Plus was effective against MRSA with a 3-log reduction, but not against P. aeruginosa, S. marcescens and S. aureus. When tested against P. aeruginosa, S. marcescens and S. aureus, 2.7-, 2.6- and 1.7-log reductions were observed respectively (Fig. 1 and Table 2), which is considered ineffective according to ISO Stand-Alone Test primary acceptance criteria [9]. Notably, the locally formulated solutions: Kontex-CLS (i.e., Kontex Clean, Kontex Normal, Kontex Multisol extra+ and Kontex Soak) did not exhibit any bactericidal activity against any of the bacterial
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R. Siddiqui et al. / Contact Lens & Anterior Eye 38 (2015) 34–38
Table 1 Contact lens solutions tested in the present study together with their ingredients and manufacturers’ minimum recommended disinfection time. Solution
Type
Ingredients
Minimum recommended disinfection time
Manufacturer
ReNu MultiPlus
Multi-purpose solution
4h
Bausch & Lomb, Greenville, USA.
DuraPlus
Multi-purpose solution
HYDRANATE® (hydroxyalkylphosphonate), boric acid, edetate disodium, POLOXAMINE, sodium borate and sodium chloride; preserved with DYMEDTM (polyaminopropyl biguanide) 0.0001% Edetate Disodium, Poloxamine, Sodium Chloride and Aquahydrate TM ; preserved with OPTIMED (polyaminopropyl biguanide). Contains no chlorhexidine, no thimerosal and no sorbic acid.
4h
Ultimate Plus
Multi-purpose solution
Polyhexamethylene biguanide, tromethamine, tyloxapol, hydroxy propyl methylcellulose (HPMC) and edetate disodium.
4h
OptiFree Express
Multi-purpose disinfecting solution
6h
Kontex Clean + Kontex Multisol extra+
Soft contact lens daily cleaning solution for sensitive eyes Soft contact lens daily cleaning, soaking, storing and cold disinfecting solution for sensitive eyes
Sodium chloride, sorbitol, edetate disodium, boric acid, aminomethyl propanol, citrate and tetronic® (polidronium chloride) 0.001% and ALDOX® (myristamidopropyl dimethylamine) 0.0005% EDTA 0.2% soft surfact. 8 polymer WS 2% in preserved isotonic solution + EDTA 0.01% citrate, poloxamer WS, S. borate, Naci, SRFACT X, HPG 0.0005% (TN) preservative. EDTA 0.2% softsurfact. 8 polymer WS 2% in preserved isotonic solution + EDTA 0.01% citrate, poloxamer WS, S. borate, Naci, SRFACT X, HPG 0.0005% (TN) preservative + borsaure M61.83 g/mol extra pure DAB, thimerosal 0.001 mg/mL, ethylenediamine tetra-A sodium salt 1 mg/mL, HPG (high preservative grades). EDTA 0.2% softsurfact. 8 polymer WS 2% in preserved isotonic solution + Isotonic buffered saline solution + EDTA 0.01% citrate, poloxamer WS, S. borate, Naci, SRFACT X, HPG 0.0005% (TN) preservative. EDTA 0.2% softsurfact. 8 polymer WS 2% in preserved isotonic solution + isotonic buffered saline solution + borsaure M61.83 g/mol extra pure DAB, thimerosal 0.001 mg/mL, ethylenediamine tetra-A sodium salt 1 mg/mL, HPG (high preservative grades).
SIPIC INTERNATIONAL Texas, U.S.A (International formulation, manufactured locally) ELKO Organization (Pvt.) Ltd. Switzerland (International formulation, manufactured locally in Karachi, Pakistan). Alcon Laboratories, Inc. Fort Worth, USA.
Kontex Clean + Kontex Multisol extra+ + Kontex Soak
Kontex Clean + Kontex Normal + Kontex Multisol extra+ Kontex Clean + Kontex Normal + Kontex Soak
Soft contact lens daily cleaning, rinsing and storing saline solution for sensitive eyes Soft contact lens daily cleaning, rinsing, soaking, storing and cold disinfecting solution
pathogens tested (Fig. 1 and Table 2). When bacteria treated with Kontex-CLS were re-inoculated on to nutrient agar plates, viable bacteria emerged in all cases and no reduction in the number of viable organisms was observed (Fig. 1 and Table 2). For CL disinfection solutions: Kontex Clean + Kontex Normal + Kontex Soak, 0-, 0.4-, 0- and 0-log reduction was observed against of P. aeruginosa,
30 s + quick rinse + soaking for 4h 30 s + quick rinse + soaking for 4h
KONTEX Corp. Laboratories, Karachi, Pakistan. KONTEX Corp. Laboratories, Karachi, Pakistan.
30 sec + quick rinse + soaking for 4h
KONTEX Corp. Laboratories, Karachi, Pakistan.
30 s + quick rinse + soaking for 4h
KONTEX Corp. Laboratories, Karachi, Pakistan.
S. marcescens, S. aureus and MRSA respectively when tested for the manufacturers’ recommended disinfection time. The combination of Kontex Clean + Kontex Normal + Kontex Multisol Extra revealed 0-, 0.4-, 0.03- and 0-log reductions when tested against P. aeruginosa, S. marcescens, S. aureus and MRSA respectively. Similarly when the combination of Kontex Clean + Kontex Multisol
Fig. 1. The efficacy of contact lens solutions against Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus and Methicillin-resistant Staphylococcus aureus. Eight different types of contact lens solutions were tested to determine their effectiveness against different bacteria according to manufacturer’s minimum recommended disinfection time as described in Materials and Section 2. Phosphate buffered saline was used as a control. White bars indicate P. aeruginosa, light grey bars indicate S. marcescens, dark grey bars indicate S. aureus and black bars indicate MRSA, treated with different contact lens solutions. The results are representative of three individual experiments performed in duplicate. The data are presented as the mean ± standard error.
R. Siddiqui et al. / Contact Lens & Anterior Eye 38 (2015) 34–38
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Table 2 The mean-log reduction of microbes tested, post-exposure of contact lens disinfectant solution as per manufacturers’ minimum recommended disinfection time. Contact lens solutions
ReNu MultiPlus DuraPlus Ultimate Plus OptiFree Express Kontex Clean + Kontex Normal + Kontex Soak Kontex Clean + Kontex Normal + Kontex Multisol Extra Kontex Clean + Kontex Multisol Extra + Kontex Soak Kontex Clean + Kontex Multisol Extra
Log reduction (standard deviation) P. aeruginosa
S. marcescens
S. aureus
MRSA
F. solani
5.13 (0.103) 5.13 (0.115) 2.72 (0.08) 5.13 (0.23) −0.24 (0.01) −0.36 (0.02) −0.43 (0.006) −0.36 (0.07)
5.76 (0.112) 5.76 (0.15) 2.61 (0.1) 5.76 (0.151) 0.38 (0.1) 0.35 (0.01) 0.20 (0.1) 0.27 (0.03)
4.41 (0.1) 4.41 (0.117) 1.74 (0.04) 4.41 (0.183) −0.07 (0.008) 0.03 (0.04) 0.02 (0.01) −0.05 (0)
4.56 (0.101) 4.56 (0.1) 4.56 (0.153) 4.56 (0.114) −0.13 (0.03) −0.04 (0.004) −0.12 (0.005) −0.15 (0.004)
5.79 (0.165) 5.79 (0.101) 2.71 (0.08) 5.79 (0.187) 0.34 (0.07) 0.32 (0.1) −0.10 (0.02) −0.05 (0.02)
Fig. 2. The efficacy of contact lens solutions against Fusarium solani. F. solani (106 conidia) were incubated with different contact lens solutions at room temperature for manufacturer’s minimum recommended disinfection time. Following this, conidia were plated onto the potato dextrose agar plates. The results represent the mean ± standard error of three independent experiments performed in duplicate.
Extra + Kontex Soak was used, 0-, 0.2-, 0.02- and 0-log reduction was observed against P. aeruginosa, S. marcescens, S. aureus and MRSA. The combination of Kontex Clean + Kontex Multisol Extra showed 0-, 0.3-, 0- and 0-log reductions when tested against P. aeruginosa, S. marcescens, S. aureus and MRSA respectively (Fig. 1 and Table 2). 3.2. Fungicidal and amoebicidal properties of contact lens disinfection solutions against F. solani
criteria [9]. However other CL disinfection solutions: Kontex Clean, Kontex Normal, Kontex Multisol extra+ and Kontex Soak were completely ineffective against fungal spores and no fungicidal effects were observed (Fig. 2 and Table 2). As expected, none of the CL disinfection solutions tested destroyed A. castellanii cysts as per manufacturers’ instructions (Fig. 3). Upon inoculation in the growth medium, cysts re-emerged as viable trophozoites (data not shown). 4. Discussion
To determine the fungicidal effects of CL disinfection solutions on F. solani, assays were performed by inoculating 106 conidiospores in 10 mL of CL disinfection solution. Of the eight CL disinfection solutions tested, the results revealed that ReNu MultiPlus, DuraPlus, Ultimate Plus and OptiFree Express were able to exhibit more than 1-log reduction in the number of viable fungal spores (Fig. 2 and Table 2). The cidal properties of ReNu MultiPlus, DuraPlus and OptiFree Express were most effective as no viable fungal colonies were seen on PDA, even after 5 days of incubation. In the case of Ultimate Plus, a 1.6-log reduction was observed which is effective according to ISO Stand-Alone primary acceptance
Contact lens wear is the most common risk factor in the development of microbial keratitis [14]. Studies to evaluate the efficacy of various types of CL disinfection solutions have been performed worldwide [15–19], whereas limited studies have been carried out for developing countries. This is particularly important as contact lens solutions are imported as well as formulated locally, and exposed to variable temperatures during storage or shipment and must be tested for their effectiveness. An increase in the incidence of microbial keratitis associated with the contact lens use at the Aga Khan University Hospital led us to hypothesize
Fig. 3. The efficacy of contact lens solutions against A. castellanii. Briefly, A. castellanii cysts (5 × 104 ) were incubated with different contact lens solutions at room temperature for manufacturer’s minimum recommended disinfection time. For controls, cysts were treated with PBS (negative control) and 1% SDS (amoebicidal effects). Next, cysts were enumerated using a haemocytometer as well as plated using growth medium (PYG). Note that none of the solutions tested had any effect on A. castellanii cysts. The results represent the mean ± standard error of three independent experiments performed in duplicate.
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R. Siddiqui et al. / Contact Lens & Anterior Eye 38 (2015) 34–38
ineffectiveness of the disinfecting solutions. Recently, the antiamoebic effects of commonly marketed CL disinfection solutions in Pakistan were tested. Astonishingly, none of the CL disinfection solutions tested had any cysticidal effects against A. castellanii keratitis isolate of the T4 genotype. Here, the efficacy of commonly marketed CL disinfection solutions against a range of bacterial and fungal pathogens was investigated. Of the eight tested solutions, ReNu MultiPlus, DuraPlus and OptiFree Express were found to be effective against all tested organisms including bacteria and fungi. On the other hand, Ultimate Plus failed to bring more than 3-log reduction in case of bacteria, including P. aeruginosa, S. marcescens and S. aureus. To our surprise, none of the locally formulated CL disinfection solutions: Kontex-CLS (Kontex Clean, Kontex Normal, Kontex Multisol extra+ and Kontex Soak) had any bactericidal and/or fungicidal effect against the corneal pathogens tested, which is a major concern for CL wearers. Additionally, none of the CL disinfection solutions were able to destroy A. castellanii cysts, which is consistent with previous studies [5]. The lack of efficacy of commonly marketed CL disinfection solutions in developing countries like Pakistan is a serious concern and suggests prevalence of microbial keratitis. As the locally formulated CL disinfection solutions are considerably cheaper to purchase, these findings may explain an increase in the number of cases of microbial keratitis (personal communication with the consultant ophthalmologists at the Aga Khan University Hospital, Karachi) suggesting the need for increased awareness. However, the actual burden of microbial keratitis remains unknown and requires further investigation. Notably, some of the known brands appeared ineffective in eradicating corneal pathogens. Previous studies have shown that the efficacy of CL disinfection solutions is affected by time and temperature of storage [20]. This may explain ineffectiveness of CL disinfection solutions tested in the present study, in addition possibly due to, lack of quality control during the manufacturing process or during importing, shipping and transportation of CL disinfection solutions and exposure to high temperatures. Manufacturers should consider this to safeguard the ocular health of contact lens wearers. Such ineffective CL disinfection solutions pose a serious risk to contact lens wearers and to the wider community. Additionally, of concern is the fact that these solutions can be purchased without any instructions given to the consumer, majority of who are unaware of the associated risks or they are teenagers in the case of cosmetic CL users. Moreover CL for cosmetic as well as corrective use are routinely available over the counter in developing countries such as Pakistan without any instructions of proper hygiene to handle CL given to the users. A study conducted at Aga Khan University, Karachi, reported that CL related corneal ulcers are resulting in serious visual impairment especially in women and many of the devastating outcomes of corneal infections are accredited to the use of contact lenses [21]. Overall, ineffective CL disinfection solutions present a major risk to public health; these findings are of great concern for health officials and to the manufacturers of the CL disinfection solutions. Travellers to developing countries who are CL wearers need to be aware of the associated risks when using any locally manufactured CL solutions. As well as being aware that the efficacy of solutions is affected by temperature. It is imperative that urgent intervention measures are implemented to address this issue.
Conflicts of interest None to declare. Acknowledgment The authors are grateful for the kind support provided by the Aga Khan University, Pakistan. References [1] Lam DSC, Houang E, Fan DSP, Lyon D, Seal D, Wong E, et al. Hong Kong Microbial Keratitis Study Group. Incidence and risk factors for microbial keratitis in Hong Kong: comparison with Europe and North America. Eye 2002;16:608–18. [2] Cheng KH, Leung SL, Hoekman HW, Beekhuis WH, Mulder PGH, Geerards AJM, et al. Incidence of contact-lens-associated microbial keratitis and its related morbidity. Lancet 1999;354(9174):181–5. [3] Poggio EC, Glynn RJ, Schein OD, Seddon JM, Shannon MJ, Scardino VA, et al. The incidence of ulcerative keratitis among users of daily-wear and extended-wear soft contact lenses. N Engl J Med 1989;321:779–83. [4] Mukherjee PK, Chandra J, Yu C, Sun Y, Pearlman E, Ghannoum MA. Characterization of Fusarium keratitis outbreak isolates: contribution of biofilms to antimicrobial resistance and pathogenesis. Immunol Microbiol 2012;55:4450–7. [5] Lakhundi S, Khan NA, Siddiqui R. Inefficacy of marketed contact lens disinfection solutions against keratitis-causing Acanthamoeba castellanii belonging to the T4 genotype. Exp Parasitol 2014;141:122–8. [6] Iqbal J, Sagheer M, Ahmed N, Siddiqui R, Khan NA. Culturable aerobic and facultative anaerobic intestinal bacterial flora of Black Cobra (Naja sp.). ISRN Vet Sci 2014 [article ID 878479] http://www.hindawi.com/journals/isrn/ 2014/878479/ [7] Siddiqui R, Sagheer M, Khan NA. Prevalence of Acanthamoeba and superbugs in a clinical setting: coincidence or hyperparasitism. Parasitol Res 2013;112:1349–51. [8] Cardas M, Khan NA, Alsam S. Staphylococcus aureus exhibit similarities in their interactions with Acanthamoeba and ThP1 macrophage-like cells. Exp Parasitol 2012;132:513–8. [9] International Standards Organization. ISO 14729 Ophthalmic optics – contact lens care products – microbiological requirements and test methods for products and regimens for hygienic management of contact lenses. Geneva, Switzerland: ISO; 2001. [10] Rosenthal RA, Sutton SW, Schlech BA. Review of standard for evaluating the effectiveness of contact lens disinfectants. PDAJ Pharm Sci Technol 2002;56:37–50. [11] Retuerto MA, Szczotka-Flynn L, Ho D, Mukherjee P, Ghannoum MA. Efficacy of care solutions against contact lens-associated Fusarium biofilms. Optom Vis Sci 2012;89:382–91. [12] Aqeel Y, Siddiqui R, Khan NA. Anti-Acanthamoebic properties of resveratrol and de-methoxy curcumin. Exp Parasitol 2012;132:519–23. [13] Dudley R, Jarroll EL, Khan NA. Carbohydrate analysis of Acanthamoeba castellanii. Exp Parasitol 2009;122:338–43. [14] Patel A, Hammersmith K. Contact lens-related microbial keratitis: recent outbreaks. Curr Opin Opthalmol 2008;19:302–6. [15] Borazjani RN, Kilvington S. Efficacy of multipurpose solutions against Acanthamoeba species. Contact Lens Anter Eye 2005;28:169–75. [16] Centers for Disease Control and Prevention. Acanthamoeba keratitis multiple states 2005–2007. MMWR: Morb Mortal Wkly Rep 2007;56:1–3. [17] Hughes R, Kilvington S. Comparision of hydrogen peroxide contact lens disinfection systems and solutions against Acanthamoeba polyphaga. Antimicrob Agents Chemother 2001;45:2038–43. [18] Miller MJ, Callahan DE, McGrath D, Manchester R, Norton SE. Disinfection efficacy of contact lens care solutions against ocular pathogens. CLAO J 2001;27:16–22. [19] Kobayashi T, Gibbon L, Mito T, Shiraishi A, Uno T, Ohashi Y. Efficacy of commercial soft contact lens disinfectant solutions against Acanthamoeba. Jpn J Ophthalmol 2011;55:547–57. [20] Leung P, Boost MV, Cho P. Effect of storage temperatures and time on the efficacy of multipurpose solutions for contact lenses. Ophthalmic Physiol Opt 2004;24:218–24. [21] Chaudhry TA, Sarfraz S, Khan QA, Ahmad K. Contact lens-related visual loss—a case series from Karachi. Pak Med Assoc 2011;61:1246–8.