EVALUATION OF THE EFFICACY OF ANTIMICROBIAL ACTIVITY OF CONTACT LENS CARE SOLUTION

Contact lens care solutions are necessary for proper hygiene of contact lenses. Numerous microorganisms can be transmitted through them, leading to serious eye infections, while blindness can occur in particularly severe cases. A direct link between ocular infections and contact lens care solutions has been proven. Commercial preparations must meet a number of safety and antimicrobial effi cacy requirements. Th is paper presents examinations of antimicrobial effi cacy of commercial preparation called LENS CARE. Th e examinations were carried out according to ISO 14729:2012 standard Stand-alone test. It has been found that commercial preparation LENS CARE within proscribed contact lens care regimen of 8h meets the primary criteria of the Stand-alone test for Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans with the results of 3.4, 3.6 and 1.0 log10 reductions, respectively. If used in accordance with the manufacturer’s instructions, the tested preparation has the desired antimicrobial eff ect against the tested pathogens.


INTRODUCTION
Contact lenses are medical devices that make life much easier for people who need vision correction, they are applied directly to the cornea and can aff ect eye health. Microbial keratitis -corneal infection is a sight-threating emergent ophthalmic disease. Th e disease is a treatable infection but can lead to a number of complications such as corneal perforation, opacifi cation, endophthalmitis (Jin et al., 2017). Th e occurrence of microbial keratitis caused by Pseudomonas aeruginosa, Staphylococcus aureus or Fusarium spp. linked with contact lenses has been directly proved (Jalbert et al., 2000;Ma et al., 2009).
Proper use of contact lenses and their care ensures eye health. Wearing lenses during the night, wearing them for multiple days, improper cleaning and disinfection, their improper storage, poor hygiene of lens storage box, and poor personal hygiene of patients can lead to eye infections (Agi et al., 2021). A great risk factor for the occurrence of infections is the sterility and effi ciency of lens care solutions. Th e outbreak of fungal keratitis occurred during 2005 and 2006 in the USA, Hong Kong and Singapore. More than 200 people became ill and the infection occurred only in people who wore contact lenses and used lens care solution "B&L ReNu with MoistureLock solution". During the epidemiological investigation, it was determined that the product was sterile, leading to a conclusion that weak antimicrobial activity of the product in combination with poor hygienic practices of the patients caused the infection. It is also believed that storage conditions aft er opening of the solution led to a signifi cant reduction in its antimicrobial activity, temperature and number of days of storing the open product (Ma et al., 2009). It is necessary to prove that lens care solutions are suitable for intended use, i.e. it is necessary to prove that the product has an adequate biocidal eff ect. In the case of agents intended for cleaning and disinfection of contact lenses, it is necessary to prove that the level of antimicrobial activity is appropriate, i.e. that the contact lens care regimen is designed to meet at least minimal performance (secondary) criteria of the Stand-alone test -ISO 14729:2012. Th e criteria are shown in Table 1. Primary criteria of 3.0 log reductions means that mean microorganism reduction is at least 99.9% and secondary criteria of 1.0 log reduction means that mean microorganism reduction is at least 90%.
Th e paper presents the results of testing the effi ciency of the commercial preparation LENS CARE during the contact lens care regimen (8h).

Product
Th e paper presents the results of examination of a commercial preparation called LENS CARE, which is intended for washing, removing proteins and lipids, disinfecting, moisturizing and storing all types of soft contact lenses. Th e preparation is sterile on the basis of 0.0001% polyhexanide. Th ree product lots were examined (Lot 1, Lot 2, and Lot 3). Manufacturer recommends contact lens care regimen of 8h.

Methods
Th e tests were performed according to the procedure described in SRPS EN ISO 14729: 2012 Ophthalmic optics -Contact lens care products -Microbiological requirements and test methods for products and regimens for hygienic management of contact lenses and European Pharmacopeia 8.0. Stand-alone test is inoculum challenge test. Th is test challenges a disinfecting product with a standard inoculum of microorganisms and establishes the extent of their viability loss (log of number of decimal reductions) at recommended contact lens care regimen.
Th e following challenge organisms were used in the tests -reference microorganisms: S. aureus ATCC 25923, P. aeruginosa ATCC 9027 and C. albicans ATCC 10231. Th e microorganisms were grown on TSA (Trypticase Soy Agar) at 30 -35 °C for 24 h (bacteria) and on SAB (Sabouraud agar) 20 -25 °C for 24 -48 h (C. albicans). Stock inoculums in peptone salt solutions were made from reference microorganisms and they were assigned total aerobic bacteria/ mold count (TAMC/TYMC) and then they were added to the LENS CARE solution in order to achieve the fi nal concentration of 5 log cfu/mL LENS CARE. Th e total count of challenge organisms was enumerated aft er 8 h. Th ree lots of products were tested, each challenge organism was tested in three samples and the determination of the number of microorganisms was performed in duplicate by seeding on TSA/SAB media. Th ese media were incubated at 30 -35 °C for 72 h (TSA) and at 20-25 °C for 5 days (SAB).
Total count of challenge organisms is shown according to log10 base. Descriptive statistics and p value was calculated using IBM SPSS Statistics 20 (IBM, Armonk, NY, USA). Th e results of the statistical tests were considered signifi cant for p < 0.05.

RESULTS AND DISCUSION
In the past , several disinfectants were used for disinfection of contact lenses, for example fi rst unpreserved saline and then disinfectants. Nowadays, all in one multipurpose product is mainly used for cleaning, disinfection and rewet in one use. Modern preparations are usually based on polyhexamethylene biguanide (PHMB) with polyaminopropyl biguanine (ReNu) or polyquaternium-1 with myristamidopropyl dimethylamine (Opti-Free), while some preparations contain only one biocide polyexanid or polyhexamethylene biguanide Th e decline in antimicrobial activity in the presence of lenses is one of the reasons why the requirements for the Stand-alone test (primary criteria) are stricter compared to the Regimen test (secondary criteria), where only one decimal reduction is suffi cient (Table 1.). Th e results of the LENS CARE solution effi ciency test are shown in Table 2 expressed as a number of decimal reductions aft er the contact lens care regimen (8h). ТAMC/TYMC vallues are expressed an average vallues of duplicate enumeration. Commercial lens care solutions are eff ective against most major eye pathogens. Antimicrobial activity depends on the type and amount of biocide contained in the preparation, and also on the method of use (Zhu et al., 2011;Gabriel et al. 2018).
S. aureus is a pathogen that can lead to eye infection, it is most commonly transmitted by hands. However, eye infections caused by the use of contact lenses have also been proven (Jalbert et al., 2000). S. aureus has the ability to form a biofi lm on various surfaces and thus signifi cantly reduces the eff ectiveness of detergents and disinfectants. log reductions. Although they used the same commercial preparation (Opti-Free Express), the same recommended time (6h) and the same test (Regimen), there were diff erences in the results. A possible explanation could be the diff erences in the contact lenses on which the test was performed or the diff erences in the design and implementation of the study itself.
Keratitis caused by P. aeruginosa is the most common type of infection transmitted through contact lenses (Agi et al., 2021). Corneal infection can develop in just a few hours (Wilson et al., 1981), so it is essential that contact lens care products are eff ective against this microorganism. In relation to antimicrobial activity against P. aeruginosa, LENS CARE is slightly less eff ective C. albicans can cause fungal keratitis, although somewhat less frequently (14%) compared to Fusarium species (41%) (Iyer et al., 2006). Candida eye infections usually occur either aft er trauma or aft er the use of corticosteroids, and it is more common in colder climates. Still, the main risk factor for all fungal keratitis is wearing contact lenses (Iyer et al., 2006, Imamura et al, 2008. Transmission of C. albicans through contact lenses is facilitated by their ability to form biofi lms (Imamura et al., 2008). Th e biofi lm that Candida forms is tightly bound to hydrogel and silicone contact lenses, so the eff ect of lens care solutions is much weaker against Candida compared to bacteria (Zhu et al., 2011). Th e eff ect of LENS CARE contact lens care products achieves at least one decimal reduction of C. albicans aft er recommended time, which is statistically signifi cantly less eff ective than challenge bacteria but still meets the primary criteria of the Standalone test. Similar results were obtained by Imamura et al (2008) for ReNu Moisture Loc and ReNu Multi Plus, while Gabriel et al. (2018) found that there is less than one decimal reduction for the ReNu Fresh preparation. Preparations based on polyquaternium and myristamidopropyl dimethylamine have much higher antimicrobial activity (1.5-4.5 log reductions) but only on the stand-alone test while their activity drops below 1 log reduction in the presence of lenses (Gabriel et al., 2018).
Th ere are no statistically signifi cant diff erences between the eff ect of the LENS CARE solution on S. aureus compared to P. aeruginosa (p = 1.0), but the number of decimal reductions of C. albicans is signifi cantly lower than S. aureus (p = 0.00003) and P. aeruginosa (p = 0.00001).

CONCLUSION
LENS CARE solution is suffi ciently eff ective and meets the requirements of the Stand-alone test in terms of antimicrobial effi cacy against to S. aureus, P. aeruginosa and C. albicans at the recommended time of 8 h. In order for the preparation to show its maximum effi ciency, it is necessary to follow the labelling instructions of the manufacturers.

Author's Contribution
Th is paper was written as a review paper which dealt with a series of papers in the fi eld of microbial quality of pharmaceutical products. All authors of this paper have participated in the writing of this paper and previous research. Writing paper JP and RR, examinations JP and IS, and data processing JP and NV.