|Year : 1994 | Volume
| Issue : 2 | Page : 65-70
Sterility and the disinfection potential of Indian contact lens solutions
Usha Gopinathan1, Savitri Sharma1, Shobha Boghani2, Gullapalli N Rao3
1 Sri Devchand Nagardas Jhaveri Microbiology Centre, Hyderabad, India
2 Bausch & Lomb Contact Lens Centre, L.V. Prasad Eye Institute, Hyderabad, India
3 Sri Devchand Nagardas Jhaveri Microbiology Centre,& Bausch & Lomb Contact Lens Centre, L.V. Prasad Eye Institute, Hyderabad, India
L.V. Prasad Eye Institute, Road No. 2, Banjara Hills, Hyderabad 500 034
Source of Support: None, Conflict of Interest: None
Ocular infection associated with microbial contamination of contact lens care products is a major problem in contact lens wearers. The sterility and the antimicrobial activity of contact lens care systems reflect their suitability for disinfection of contact lenses. These factors remain to be evaluated for the various newer contact lens care products manufactured in India. In this study, 35 bottles of contact lens solutions marketed by different manufacturing units in India were tested for sterility. Seven solutions were tested for antimicrobial effectiveness employing the D value method of analysis. The D value is defined as the time required to reduce a population of organisms by 90% (one log unit). A standard inoculum of the ocular isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Serratia marcescens, Aspergillus fumigatus, Fusarium solani, and Acanthamoeba castellanii were used as challenge organisms. Bacterial contamination was detected in 20 (57.1%) solution bottles and none yielded fungus or Acanthamoeba. Pseudomonas species were the most commonly encountered contaminant (11/20; 55%). Only sterile solutions were analyzed for antimicrobial activity. D values ranging between 12 and 20 minutes were demonstrated by six of the seven solutions against bacterial challenge. Good antifungal activity was noticed in five solutions against Fusarium solani though results varied with Aspergillus flavus and Candida albicans. All solutions were adequately effective against Acanthamoeba.
Keywords: Sterility - Antimicrobial efficacy - Contact lens care solutions - D value
|How to cite this article:|
Gopinathan U, Sharma S, Boghani S, Rao GN. Sterility and the disinfection potential of Indian contact lens solutions. Indian J Ophthalmol 1994;42:65-70
The widespread use of contact lenses has resulted in an increased incidence of microbial keratitis.  Among the various factors reported as being responsible for contact lens-related ocular infections, microbjal contamination of lens care products is a frequent observation . ,, Wilson et al sub have established the serologic and physiologic correlation of the strains obtained from the ocular surfaces and the eye care solutions used by patients with corneal ulcers. Studies have demonstrated a high frequency of contamination of the lens care systems even in asymptomatic contact lens wearers,  thereby questioning the safety of its use by the susceptible population. Donzis et al,  who examined homemade saline and commercial contact lens solutions used by 100 asymptomatic patients, found 100% (12/12) and 13% (16/126) microbial contamination in the solutions respectively.
With the growing concern over the potential hazards to contact lens wearers, evaluation of the disinfecting activity of the contact lens products has been an interesting area of research.  Such studies were mainly aimed at achieving the best desirable characteristics of the disinfecting systems. With the increase in use of contact lenses in India, a variety of contact lens care products have been introduced into the local market for the routine maintenance of contact lenses. Poor antifungal activity of Indian contact lens solutions has been described previously, where the authors have also detected bacterial contamination of fresh unused bottles. 
The sterility and the disinfection potential of various new products remain to be evaluated. The present study was conducted to investigate the safety and efficacy of the contact lens solutions manufactured in India.
| Materials and methods|| |
Thirty-five bottles of contact lens care solutions with various brand names and batch numbers manufactured by eight different companies in India were evaluated for microbial contamination. All products were previously unopened and unused. They were used for investigation before the expiry dates mentioned by the manufacturer. Solutions used as cleaning, rinsing and soaking agents for soft and rigid gas permeable lenses were included in the study. Most solutions contained either one or two preservatives such as chlorhexidine gluconate, thimerosal, and sorbic acid. The bottles were first examined to check if they were marketed with opened or closed nozzles. After coding, the solutions were transferred into sterile test tubes using aseptic techniques. One hundred microlitre aliquots of the solutions were used for inoculating various media. Media were incubated at varying atmospheric conditions [Table - 1] and examined for growth. The number of colony forming units of the bacterial growth was determined and the isolates were identified by standard microbiological methods. Of the 20 contaminated solutions, twelve were randomly rechecked using fresh bottles with same batch numbers, in order to find out whether or not all products of the same batch were contaminated with the same organisms.
Seven solutions from five different manufacturers that were tested twice and found free of contamination were subjected to antimicrobial efficacy testing by the D value method of analysis described by Lowe et al.  The D value is the time required to reduce a population of organisms by 90% (one log unit). Ocular isolates of Staphylococcus aureus,Pseudomonas aeruginosa, Serratia marcescens, Aspergillus fumigatus, Fusarium solani, and Acanthamoeba castellanii,were used as challenge organisms.
The bacteria and yeasts were grown in trypticase soy broth for 24 hours in a shaking water bath (75 rpm) maintained at 35°C. Normal saline was used to harvest the cells to get a suspension of 10 6 and 10 8 organisms per millilitre, respectively, as determined by spectrophotometry. The microbial counts were verified by culturing serial dilutions of the overnight incubated trypticase soy broth.
The moulds were grown on potato dextrose agar at 27°C for 10 days and the spore density was adjusted to 10 8 spores per millilitre with normal saline, using spectrophotometry and serial dilutions.
Acanthamoeba castellanii was grown for 4 days at 35°C on nonnutrient agar seeded with Escherichia coil. The inoculum was standardized by suspending cysts and trophozoites in saline at pH 7.2 and adjusting the count using haemocytometer to 1 X 10 8 organisms per millilitre with a 8:2 ratio of cysts to trophozoites.
One millilitre each of the standard bacterial and fungal suspension was transferred to 9 ml of the test solutions and incubated at room temperature. For testing antiamoebic efficacy, 0.1 ml of Acanthamoeba suspension was added to 5 ml of the contact lens solution and incubated at room temperature.
At each predetermined sampling time, (0, 15, 30,45,60, and 75 minutes for bacteria and 0,30,60,90, and 120 minutes for fungi and Acanthamoeba) the viable counts of the organisms were determined by serial dilution as colony forming units per millilitre (cfu/mL). These were plotted against the preselected time points to calculate the D value by the method of "D value between intervals" .  Between different time intervals the D values were measured for the entire contact time and an average value of these figures was taken as the D value of a solution for the respective microbial challenge.
| Results|| |
Of the 35 solutions tested for sterility, 20 (57%) manufactured by eight different companies demonstrated bacterial contamination. Of the 20 contaminated solutions, 12 were rechecked for sterility. In 10 solutions the same organisms were detected, one solution grew a different bacteria, and the other turned out to be sterile. None were contaminated with fungus or Acanthamoeba. Multiple organisms were detected in seven solutions. Of the eight brands of solutions tested, products of only two brands were consistently sterile.
Of the 35 solutions analyzed in this study, 21 were marketed with closed nozzle. The open and the closed nozzle bottles had a contamination rate of 8/14 (57%) and 12/21 (57%), respectively. The bacterial species and their rate of isolation from the solutions is shown in [Table - 2]. Pseudomonas species were the most predominant organism. Ninety-five percent (19/20) of the contaminated solutions were associated with gramnegative rods. The level of contamination by each bacterial species as determined by colony count method is detailed in [Table - 2]. The cell densities in solutions contaminated with gram-negative bacteria ranged between 20 and 1.2X10 8sub cfu/mL and with gram-positive bacteria between 40 and 4 X 10 5sub cfu/mL.
The D values of the seven test solutions for the selected bacteria are illustrated in the Figure. Except solution 7 that expressed a D value of 3.6 minutes for Pseudomonas aeruginosa, all other solutions exhibited D values between 12 and 20 minutes for the three bacterial species tested. The data in [Table - 3] shows the D values obtained for the three fungal species. Over 90 % reduction of the challenge microorganisms could not be achieved within 120 minutes with solutions 1 and 2 for Candida albicans. However, the D values ranged between 10.1 and 67.8 minutes for the same organism for solutions 3 to 7. Solution 5 demonstrated poor antifungal efficacy against Aspergillus flavus and Fusarium solani. At 0 hour incubation itself, solutions 1 to 4 and 7 were found very effective against Fusarium solani revealing no fungal growth on culturing the solution. Similarly, solutions 1,2 and 6 were effective against Acanthamoeba castellanii at 0 hour. The remaining solutions were effective against Acanthamoeba castellanii after 30 minutes contact. All solutions gave similar results when repeated for antimicrobial efficacy with the challenge microorganisms.
| Discussion|| |
Infectious keratitis in contact lens wearers is multifactorial. Microbial contamination of contact lenses, lens cases, and contact lens solutions has been identified as factors predisposing to microbial keratitis. In the present study a high degree of microbial contamination of the indigenously manufactured lens care solutions has been observed, with the contamination rate being 57%. It is evident from our observation that the solutions were not contaminated after usage. The bottle caps were opened only at the time of investigation. Donzis et all found a greater contamination rate in solutions that were used for a longer period than in those opened and used for a shorter duration. When compared to contact lens care systems used for daily wear lenses, a higher incidence of contamination has been reported with those used for extended-wear lenses .  The evidence of microbial contamination in unused lens care solutions poses a great threat to the population of contact lens wearers in India. Furthermore, the contamination of paired solutions of the same batch with similar organisms is a reflection of poor aseptic techniques adopted during the manufacturing process by many of the manufacturers in India.
None of the bottles subjected to sterility checking revealed the presence of fungi or Acanthamoeba. They have been detected as contaminants in different brands of contact lens solutions after usage by Donzis et al.  In their study, the authors opined that bacterial or fungal contamination act as a nutrient source favouring the growth of Acanthamoeba. Hence, there is a greater possibility of Acanthamoeba appearing as a contaminant in solutions already harbouring bacteria.
Interestingly, there was no difference in the rate of contamination between open and closed nozzle bottles, indicating that solutions were contaminated prior to bottling.
The type of organisms recovered from the solutions used in our study are of great concern, the gram-negative bacteria predominating the spectrum. Gram-negative organisms have frequently been identified as agents causing ulcerative keratitis. , The endotoxin has been demonstrated in solutions contaminated with gram-negative rods  and it is also reported to cause corneal changes.  The toxins that could be present in solutions showing gram-negative bacterial contamination in the present study, can lead to ocular tissue damage and ulceration in contact lens users. The incidence of contact lens-related keratitis caused by Pseudomonas , and its prevalence in contact lens care systems , are reported to be very high. These organisms are capable of surviving for a longer period in moist environment and can retain viability within the glycocalyx biofilm.  Fifty-five percent (11/20) of the solutions evaluated for microbial contamination in this series were found to contain Pseudomonas species, eight of them being P.aeruginosa, two P.maltophila, and one an unidentified species.
Investigators have employed several methods for the estimation of antimicrobial efficacy of contact lens care solutions,  one among them being the D value method of analysis. Discrepancies in D values for the different microbial challenge have been noted in earlier reports, which could be attributed to the variation in the concentration of chemical components of contact lens care solutions, methods adopted for D value estimation, inoculum load of microbes, the strains used, and factors supporting their growth.
The results of the current experiment indicate that most solutions expressed greater disinfecting activity against Acanthamoeba castellanii when compared to bacterial and fungal challenge.
No recovery of Pseudomonas aeruginosa was observed in 4 solutions at the 75 minutes contact time, two of them being equally effective against Candida albicans and Fusarium solani, respectively at 85 minutes and 90 minutes contact. Only one brand expressed a similar activity against Staphylococcus aureus. In addition, the fungicidal activities of 5 solutions against Fusarium solani were noteworthy with no survival of spores at the 0 hour sample time itself.
When compared to the report on non-peroxide disinfection solutions manufactured abroad,  the present study reveals that a much lesser contact time is required by most solutions for a one log unit decline in the spore densities of fungal species. Our study differs from the cited study for bacterial challenge because the D values in their experiments were less than 40 seconds for different bacterial strains. Shih et al  have shown D values ranging between 8 minutes and >4 hours in their study on two fungal species. However, a relatively greater antifungal activity has been shown by hydrogen peroxide (3 % ) solutions with D values of 11 minutes and 48 minutes for Aspergillus fumigatus and Candida albicans, respectively.  Exposure times as short as 30 minutes or less was sufficient to eliminate Acanthamoeba castellanii which is in agreement with an earlier report,  where the efficacy of chlorhexidine was highlighted. Four solutions in our series contained this preservative.
In practice, where organisms have adhered or penetrated the contact lenses, the time required for various contact lens care solutions to penetrate the lens and to bring about microbial elimination should be taken into account for achieving good results. Our data clearly demonstrates that adherence to strict quality control measures during manufacture should be mandatory. The performance of D value testing of the solutions should be made mandatory as a screening procedure for considering their inclusion in further evaluation of adequate antimicrobial efficacy. Consumer education in proper contact lens care is possible only when the lens care product itself assures effective lens disinfection. Practioners should therefore be advised to take into consideration the various factors that determine a successful contact lens practice. The practitioner should be cognizant of the potential for infection and initiate necessary measures to eliminate the usage of contact lens solutions of poor quality.
This study highlights the relative laxity in quality control during manufacture and lack of proper regulatory controls to ensure safety and efficacy of most of the contact lens solutions available in the Indian market.
| Acknowledgement|| |
This study was supported by a grant from Bausch & Lomb International Division Research Programme, Rochester, New York, U.S.A.
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[Figure - 1]
[Table - 1], [Table - 2], [Table - 3]
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