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Microbial contamination of multiply used preservative-free artificial tears packed in reclosable containers
  1. M S Kim1,
  2. C Y Choi1,
  3. J M Kim1,
  4. H R Chung1,
  5. H Y Woo2
  1. 1
    Department of Ophthalmology, Sungkyunkwan University School of Medicine, Kangbuk Samsung Hospital, Seoul, Republic of Korea
  2. 2
    Department of Laboratory Medicine, Sungkyunkwan University School of Medicine, Kangbuk Samsung Hospital, Seoul, Republic of Korea
  1. Dr C Y Choi, Department of Ophthalmology, Sungkyunkwan University School of Medicine, Kangbuk Samsung Hospital, 108, Pyeongdong, Jongnogu, Seoul, Republic of Korea; sashimi0{at}naver.com

Abstract

Background/aims: To evaluate microbial contamination of multiply used preservative-free artificial tears packed in reclosable containers after daily use.

Methods: Subjects were provided with preservative-free artificial tears (Groups 1 and 2) and artificial tears containing a preservative (Group 3). After administration three times or more per 10 h, bottles were collected, and any remaining fluid in the bottles was cultured. A risk factor analysis for microbial contamination was performed by the use of univariate and multivariate analysis.

Results: A total of 242 eye-drop bottles were evaluated. Five (2.0%) of the 242 bottles had bacterial contamination. In group 1, four (3.9%) of 102 bottles were contaminated, and identified bacteria were all coagulase-negative Staphylococcus. In group 2, one (1.0%) of 105 bottles was contaminated, and it was a Gram-negative Acinetobacter species. No bottles from group 3 showed any contamination. Based on multivariate analysis, advanced age and fingertip touch were statistically significant risk factors for microbial contamination (p<0.05).

Conclusion: Preservative-free artificial tears in reclosable containers are at risk of contamination in a daily and multiple use setting, especially in patients with a poor administering technique, which is associated with fingertip touch and advanced age.

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The administration of contaminated eye-drops may lead to serious ocular infectious disease. Pseudomonas and Serratia associated serious bacterial keratitis have been reported to be transmitted by the use of contaminated eye-drops.13 Recently, artificial tears have been used as main therapy for the treatment of dry eye syndrome. Artificial tears usually contain hydrophilic polymers designed to lubricate the ocular surface and supplement the tear components to relive symptoms briefly and temporarily.

Numerous brands of artificial tears are available as unit- or multidose formulas. Multidose artificial tears usually contain preservatives to maintain sterility after opening the container. However, eye-drops containing preservatives may produce toxic or allergic reactions particularly with frequent instillation or when applied to an injured ocular surface.46 Therefore, preservative-free artificial tears have been introduced as an alternative to multidose artificial tears with preservatives. Preservative-free artificial tears have been packed in unit-dose containers to reduce the risk of microbial contamination. The unit-dose containers are recommended to be used once and are to be discarded after instillation.4 Despite a higher cost than artificial tears containing a preservative, unit-dose artificial tears have been widely used to avoid exposure to irritant preservatives.

To date, reclosable containers containing preservative-free artificial tears for extended use in the form of daily and multiple use packages have become available. Some studies have evaluated and reported microbial contamination of preservative free eye-drops with extended use.7 8 The aim of the current study was to evaluate microbial contamination of multiply used preservative-free artificial tears packed in reclosable containers after daily use.

MATERIALS AND METHODS

The current study was a prospective, non-masked and randomised trial. This study consisted of two parts. The first part was to evaluate the occurrence of microbial contamination of unpreserved artificial tears packed in reclosable containers after daily use as compared with the use of artificial tears containing preservatives. The second part of the study was challenge testing to compare the growth of micro-organisms in different preservative free artificial tears with equal concentrations of bacteria and under identical environmental conditions.

Occurrence of microbial contamination

A total of 242 bottles containing artificial tears were collected after instillation of the artificial tears in volunteers without any ocular infectious disease. Subjects in group 1 and group 2 were provided with preservative-free artificial tears with different hydrophilic polymers. Subjects in group 3, a control group, were provided with artificial tears containing a preservative. Group 1 subjects were provided a unit-dose bottle containing 0.8 ml of preservative-free 0.1% hyaluronate. Group 2 subjects were provided with an over-the-counter preparation in a unit-dose bottle containing 0.4 ml preservative-free 0.5% carboxymethylcellulose. Group 3 subjects were provided with a multidose bottle containing 5 ml of 0.1% hyaluronate preserved with 0.03% benzalkonium chloride. The bottles provided to group 1 and 2 subjects were made of a single plastic material. The closure cap was designed to be twisted off from the body of the bottle and was reclosed by plugging the cap into the tip of the body between instillations. To exclude contamination during the culture procedures, 10 cultures of artificial tears that were not given to the volunteers were performed immediately after opening the bottles in each of the three groups.

The volunteers were instructed that application of the eye-drops should be at least three times per 10 h. The subjects were instructed to cap the closure cap between applications. Ten hours after use, bottles were collected by personnel from the Department of Ophthalmology, and cultures were performed at the microbiology laboratory within 1 hour. For each bottle, a culture of the remaining solution was performed on blood agar plates (BAP) and McConkey agar plates by qualitative and quantitative methods. For qualitative measurement, the bottles were inverted, and one drop was inoculated on sterile plates. In order to measure micro-organisms quantitatively, the surface streak method was performed using a 1/1000 ml calibrated loop. The loop delivered 0.001 ml of solutions to the surface of the plate, making each resultant colony representative of 1000 colony-forming units (CFU)/ml in the original solutions. The inoculated plates were incubated at 37°C for 24 h. Colonies on the plates were counted at the end of the incubation period. All micro-organisms were identified by Gram staining and biochemical assays.

Answers to questionnaires were obtained from the participants of this study about age, sex, instillation frequency, whether the tip of the bottles touched the cilia, ocular surface or fingertip and the previous use of artificial tears. All of the volunteers gave informed consent for the experimental purpose of the used bottles and knowledge about demographics. This study was approved by the Institutional Review Board under the tenets of the Helsiniki declaration from the Ethics Committee of Kangbuk Samsung Hospital, Seoul, Korea.

Challenge testing to compare the growth of micro-organisms

To compare the growth of the micro-organisms between the different artificial tears preparations, the growth of two micro-organisms among the cultured bacteria was investigated. Two micro-organisms were subcultured on the BAP or McConkey agar plates at 37°C for 24 h. The density of suspensions containing micro-organisms was adjusted to approximately 108 CFU/ml by comparing its turbidity with a McFarland 0.5 standard. Each suspension of 0.01 ml was mixed with 10 ml of each artificial tear in an aseptic tube to make a mixture with a final concentration of approximately 105 CFU/ml. From each mixture, 0.001 ml of the drop was spread over BAP or McConkey agar plates. After streaking, incubation was performed at 37°C for 24 h, and colonies on the plates were counted and compared. A total of 20 cultures were performed with the mixtures containing various associations of each micro-organism and each artificial tears. The pH was measured with a pH meter, and the osmolality was measured with a micro-osmometer for 10 samples of artificial tears of each group before diluting the micro-organisms with the eye-drops.

Statistical analysis was performed using SPSS for Windows 15.0 to determine significant differences in the occurrence of cultured micro-organisms and the number of colony-forming units between samples from the three groups or from between group 1 and 2 samples. Risk factor analysis for microbial contamination was performed by univariate analysis with use of the χ2 test and by multivariate analysis with the use of the binary logistic regression test.

RESULTS

Occurrence of microbial contamination

Ten cultures were performed immediately after opening of the bottles in each of the three groups, and there was no growth of micro-organisms. A total of 242 eye-drop bottles were evaluated. The mean age of subjects in the three groups was 35.6 (SD 13.4) years and the mean frequency of applying eye-drops was 3.61 (0.9) times/10 h. There were no significant differences in the mean age and frequency of applying eye-drops among subjects in the three groups (p>0.05).

Five (2.0%) of the 242 bottles had bacterial contamination. In group 1, four (3.9%) of 102 bottles were contaminated. Identified organisms were all coagulase-negative Staphylococcus including two S epidermidis (28 and 32×103 CFU/ml), one S warneri (35×103 CFU/ml) and one S xylosus (3×103 CFU/ml). In group 2, one (1.0%) of 105 bottles was contaminated, and the identified organism was Gram-negative Acinetobacter baumannii (69×103 CFU/ml). No bottles in group 3 showed contamination.

There was no significant difference in the contamination rate between preserved and preservative free (p = 0.353) and between the bottles in group 1 and group 2 (p = 0.176).

Challenge testing to compare the growth of micro-organisms

Two micro-organisms—Staphylococcus epidermidis and Acinetobacter baumannii—were cultured. No growth was found in the artificial tears from group 3, and there was no significant difference in the colony counts determined between group 1 and group 2. The mean CFUs of group 1 and 2 were 59.6 (29.2) and 72.8 (28.0) (×103 CFU/ml, p = 0.153) for Staphylococcus epidermidis, and 208.2 (84.4) and 206.4 (84.8) (×103 CFU/ml, p = 0.947) for Acinetobacter baumannii. The mean pH was 6.67 (0.11) in group 1 and 6.50 (0.03) in group 2, which was a significant difference (p<0.001). The mean osmloality of the artificial tear solution was 308.30 (2.31) in group 1 and 288.40 (1.58) mOsm/l in group 2, which was also a significant difference (p<0.001).

Univariate and multivariate analysis were performed excluding group 3 artificial tears for risk factor identification. The evaluated risk factors for microbial contamination were group, age, sex, applying frequency, whether the tip of the bottles touched the cilia or ocular surface, whether the tip of the bottles touched the fingertip and previous use of artificial tears. The age of subjects who submitted contaminated artificial tears was significantly more advanced (p = 0.020), and fingertip touch to the tip of the bottle was significantly more common in contaminated bottles than in uncontaminated bottles (p = 0.050) (table 1).

Table 1 Characteristics of subjects based on microbial contamination of artificial tear solutions

Group 1 artificial tear, age over 50 years, cilia or globe touch and fingertip touch were adapted for multivariate analysis. By multivariate analysis, age over 50 years and fingertip touch were statistically significant risk factors for contamination (p<0.05). The odds ratios and significances are shown in table 2.

Table 2 Multivariate analysis of the risk factors for microbial contamination of artificial tear solutions

DISCUSSION

Manufacturers of preservative-free artificial tears usually recommend that unit-dose phials should be used once and then discarded. However, multiply used preservative-free artificial tears are often encountered in the outpatient setting. This may be due to the following reasons. First, after opening the unit phial by twisting off the tip material from the body, the tip acts as a closure cap and can be plugged into the original position. Second, the amount of artificial tears in the phials occasionally exceeds the amount that is required for one application. Third, it is often difficult to educate the patients on how to use the phials. Some patients used the phials multiply for their abundant amount despite sufficient explanation of single use. The tip designated as a cap and large amount of phial make us doubt if the phials were intended by manufacturers for multiple use. For these reasons, we were determined to evaluate contamination of multiply used unpreserved artificial tears.

In the current study, five (2.4%) of the microbial contaminations were detected in 207 reclosable containers containing daily and multiply used preservative-free artificial tears. To the best of our knowledge, there are no previous studies on the contamination rate of daily in-use preservative-free artificial tears. There have been studies evaluating contamination rates of eye-drops containing preservatives. The rate of contamination of preserved eye-drops has been reported to range from 2.2% to 34.8%,716 and this was usually higher than that of the current study. The artificial tears of this study were used for a shorter period (10 h) than eye-drops containing preservatives. The opportunities for contact with exogenous micro-organisms were relatively lower in daily use than eye-drops containing preservatives usually used for 2–4 weeks.

Among the five cultured micro-organisms, four (80%) were Gram-positive coagulase-negative Staphylococcus (CNS), and one (20%) was a Gram-negative organism. CNS is known as a commensal normal flora of the conjunctiva and skin.17 A higher occurrence of Gram-positive CNS than pathogenic Gram-negative organisms corroborates previous studies dealing with preserved eye-drops.15 16 Geyer et al reported that 69% of cultured micro-organisms were CNS in the cultured micro-organisms in a study of glaucoma eye-drops.15 In the present study, fingertip touch was a significant risk factor for microbial contamination of eye-drops as determined by multivariate analysis (table 2). This finding suggests that the contamination originated from the skin of the patient’s hand. Based on multivariate analysis, advanced age was also a significant risk factor, and this may be due to a poor technique in administering the eye-drops. Elderly subjects usually have poor vision, especially in the form of presbyopia, poor coordination and difficulty in fine grasping, which may lead to fingers touching the tip and regurgitation of the eye-drop into the bottle.

In the challenge testing, there was no growth in samples from group 3, and no significant differences in growth between groups 1 and 2. The first aim of challenge testing was to evaluate the effect of preservatives. There was no contamination case of preserved tears from group 3 in the first study, and challenge testing also showed that an adequate preservative effect may cause no growth in the group 3 tears. The second aim was to validate whether there was any growth difference between group 1 and 2 samples, which might cause a higher rate of contamination in group 1 samples, although it was not statistically significant. Despite the differing hydrophilic component, initial pH and osmolality, there was no significant difference in the growth of the organisms in the challenge testing. In addition, based on univaraite and multivariate analysis, whether the artificial tears provided to group 1 or group 2 subjects was not a significant risk factor for contamination.

In terms of the microbial culture, unlike previous studies,10 11 13 16 we obtained culture materials only from the dropping and not from the tip or cap. This was because the piece acting as a cap was not originally covering the tip of the body but connected to the tip of the body by plastic material. The tip covered by the cap was exposed before the bottle was reclosed and had a potential risk for contamination. Therefore, it may be impractical to perform a culture from an initially non-sterile tip or after a period longer than 10 h.

In conclusion, preservative-free artificial tears in reclosable containers are at risk of contamination in a daily and multiple-use setting, particularly in patients with a poor administering technique, which is associated with fingertip touch and advanced age. Contamination may cause serious ocular infections. Subjects need to be educated on the precise administering technique of unpreserved reclosable artificial tears. If cooperation is not expected from users such as older people or individuals with poor handling techniques, it may be helpful to recommend the use of single-use preservative-free artificial tears or artificial tears containing a preservative. Further studies should be performed to determine if contamination occurs in well-educated users.

REFERENCES

Footnotes

  • Competing interests: None.

  • Ethics approval: Ethics approval was provided by the Ethics Committee of Kangbuk Samsung Hospital, Seoul, Korea.

  • Patient consent: Obtained.

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