DARU Volume 12, No. 4, 2004
151
MICROBIAL CONTAMINATION OF PRESERVED OPHTHALMIC DROPS IN OUTPATIENT DEPARTMENTS:
POSSIBILITY OF AN EXTENDED PERIOD OF USE
1
MOHAMMAD REZA FAZELI,
2HOUSHANG BEHESHT NEJAD,
3HADI MEHRGAN,
1LEILA ELAHIAN
1
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy,
2Ophthalmology Division, Farabi Eye Center, Tehran University of Medical Sciences,
3Department of Pharmaceutics, Faculty of Pharmacy, Shaheed Beheshti University of Medical Sciences,
Tehran, Iran
ABSTRACT
Ocular infections may arise from topical ophthalmic medications. A standard imposed by the British Pharmaceutical Codex implies that eye drops should be discarded after 1-day use when these remedies are used in outpatient departments. In this study the bioburden rates arising from 2, 4 and 7 days’ use were evaluated and compared with those of 1 day’s use to determine whether it is possible to extend the period of use of preserved eye drops in outpatient departments.
A total of 200 eye drops were taken from outpatient departments of Farabi Eye Hospital after 1, 2, 4 and 7 days’ use and the contamination rates of the residual contents, caps and droppers were determined using conventional techniques. High biobudren rates were obtained in all the samples tested. Although the overall recorded incidences of microbial contamination in the 2 and 4-day drops were not considerably different from those of first day (P>0.01) but those of 7 days’ use were significant (P<0.01). However, when contamination rate of drop contents was taken into account there was a significant difference between 4 and 7 days’ use compared to 1-day drops. Most of the isolated organisms were either of human flora types of Gram-positive bacteria or air-borne fungi. It is concluded that the use of eye drops for outpatient practice may be extended up to 2 days; yet, care should be taken to reduce the overall contamination rates of these preparations for prevention of ocular nosocomial infections.
Keywords: Drug contamination, Ophthalmic solutions, Eye drop, Outpatient, Bacteria
INTRODUCTION
Contaminated eye drops must be postulated as a serious risk factor for ocular infections (1).
Endophthalmitis due to eyedroppers contaminated with Pseudomonas pyocyanea (2) as well as some cases of keratitis caused by Serratia contaminated eye drop caps and droppers have been reported (3). It has also been determined that 5.6% of the samples collected from hospital wards, clinics and outpatients were contaminated with viable bacteria (4). Survival of bacteria has also been found in the bottle dead spaces of eye drops (5, 6).
High contamination rates of residual contents such as 34.8% (7), 29% (8), 28% (1), 27% (9), 18% (10) and 12.9% (5) have been reported.
However, other investigations (11-14) have shown considerably lower bioburden rates (2.2%, 2.3% and 2.5% and 0.07% respectively).
Following reports on the microbial contamination of eye drops, a number of standards for safe administration of eye drops products were legislated in the British Pharmaceutical Codex
(15), which are still applied (16). According to one of these standards, eye drops used in outpatient departments should be discarded not later than one day after first opening. However, this standard seems to be an arbitrary figure and lacks adequate scientific background (17, 18). As ophthalmic medications are prepared sterile and efficient preservative systems are usually added to these products, an extended period of use of eye drops in in-patients and outpatients could lead to a substantial saving. The incidences of microbial contamination for 7 and 14 days’ use of the eye drops residues have been found to be 6.1% and 9.1% respectively, indicating no significant difference (17). The aim of this study was to compare the microbial contamination of the residual contents, droppers and caps of eye drops used by outpatient departments in Farabi Eye Hospital, Tehran University of Medical Sciences after 1, 2, 4 or 7 days of usage to assess the validity of an increased in-use period for preserve- ed eye drops opened in outpatient departments.
Correspondence: Mohammad Reza Fazeli, Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran, Email: [email protected]
MATERIALS AND METHODS Sample Collection
A total of 200 eye drops in-use were collected during September to December 2002 from outpatient departments of Farabi Eye Hospital (Ophthalmology Center of Tehran University of Medical Sciences, Tehran, Iran) after 1, 2, 4 or 7 days of use (50 samples each). Eye drop residues containing an active drug with inherent antimicrobial properties e.g. antibiotics were excluded from the study. All the collected samples were recorded for their active ingredients as well as their duration of uses. The residual contents, caps and droppers of the eye samples were immediately examined for possible microbial contamination under aseptic conditions.
Methods
The droppers were detached and immersed into Soybean-Casein Digest Broth (SCDB) and incubated at 32.5 ± 2.5 oC for 48 h. The incubated tubes were examined and cultured in Soybean- Casein Digest Agar (SCDA) and Saburouad Dextrose Agar plus Chloramphenicol (SDA+C) in the case that any microbial growth was observed.
The SCDA and SDA+C plates were incubated at 32.5 ± 2.5 oC for 48-72 h and at 22.5 ± 2.5 oC for 5-7 days, respectively. The isolates were identified using conventional microbiological techniques (19).
Sterile cotton-tip swabs were moistened with sterile saline and were rotated around the inside tip plus inside edge of the caps. The SCDA and SDA+C plates were then inoculated with separate swabs and incubated as mentioned above.
Validation of preservative inactivation under the test conditions were conducted according to the USP recommendations (20). For each eye drop container, 0.5 ml of the residual liquid was aseptically pipetted and diluted to 5 ml with sterile Soy-Casein Digest Polysorbate 20 broth (containing 4% Tween 20 and 0.5% Lecithin) to inactivate preservative.
Data analysis
In order to assess the statistical significance between the ratios, a χ2 analysis was carried out at a 5% significance level using SPSS® 10.0 (SPSS Inc., Chicago, IL, USA).
RESULTS
The entire eye drops analyzed harbored benzalkonium chloride as preservative except the artificial tears drops which had parabens instead.
All the drops had polyethylene containers. The results on the validation procedure of the neutralization method which were used were satisfactory (data not shown).
A total number of 200 eye drops with these specifications were microbiologically evaluated following 1, 2, 4 or 7 days of use. The incidence of microbial contamination of the four groups of eye drops is depicted in table 1. Although the recorded incidences of microbial contaminations were apparently greater for eye drops after 2 or 4 days of use (54% and 60% respectively) in comparison with those of 1 day’s use (44%), the differences were not statistically significant (P>0.1). A high significant difference in contamination however was observed between eye drops after 7 day’s use with regard to those after 1 day’s use (P<0.01).
Table 2 illustrates the contamination rates obtained from the residual contents, caps and droppers after 1 to 7 day’s of use. Unexpectedly, none of the droppers were found to be contaminated, while caps and residual contents showed different degrees of contamination that increased by the length of the use. The most contaminated part of the ophthalmic drops was residual contents (50%), whereas a lower contamination rate (31.5%) was obtained in eye drop caps (P<0.001). Caps and contents of the eye drops which had been used for 4 or 7 days disclosed a remarkably higher contamination rate compared with those from the first day (P<0.03), while those from 2 day’s use showed no considerable differences.
The contamination rates recovered from individual eye drops have been shown in table 3.
Tetracaine HCl, Betamethasone and the Cocktail made from Atropine sulfate 1%, Phenylephrine HCl 2.5%, Cyclopentolate HCl 1% and Tropicamide 1% (in equal proportions) at the outpatient departments had the highest overall rates of contamination.
Identities of the contaminating microorganisms are depicted in table 4. It should be noted that in many cases more than one microorganism was isolated from the eye drops. A wide range of Gram-positive bacteria and fungi were detected in the contaminated eye drops. However, most of the isolates were either of human micro-flora or saprophyte organisms.
DISCUSSION
In this study, a certain number of eye drops which were used for 1, 2, 4 or 7 days in outpatient departments of Farabi Eye Hospital were microbiologically examined. Apart from 7 day’s use of the eye drops, which appeared to be highly contaminated, no significant difference was observed between contamination rates of 2 and 4 day’s use with that of 1 day. However, the rate of isolated contaminants showed an increasing trend when the duration of the usage was extended.
When contamination of different sites of eye
Microbial contamination of ophthalmic drops
Table 1. Incidence of microbial contamination of eye drops analyzed after 1, 2, 4 and 7 day’s use.
Number of eye drops analyzed Duration of use
(Day) Contaminated Uncontaminated % Contaminated
1 22 28 44 2 27 23 54 4 30 20 60 7 35 15 70*
* Significant difference from the first day (χ2 test, P<0.01).
Table 2. Bioburden rates of the caps, droppers and the residual contents of 1, 2, 4 and 7 day’s eye drops in outpatient departments.
Duration of use
(Day) Cap contaminated/total
(%) Dropper contaminated /total
(%) Content contaminated/total (%)
1 6/50 (12) 0/50 (0) 17/50 (34)
2 11/50 (22) 0/50 (0) 26/50 (52)
4 17/50 (34)* 0/50 (0) 28/50 (56)*
7 29/50 (58)* 0/50 (0) 29/50 (58)*
* Significant difference from the first day (χ2 test, P<0.03).
Table 3. Contamination rates of the individual eye drops after 1, 2, 4 and 7 days’ use in outpatient departments.
Number of contaminated containers / total (%) Generic name
1 Day’s use 2 Days’ use 4 Days’ use 7 Days’ use Overall contamination Tetracaine Hcl 12/15 (80) 15/17 (88.2) 15/19 (78.9) 12/13 (92.3) 54/64 (84.4) Betamethasone 0/1 (0) 1/1 (100) 3/3 (100) 4/5 (80) 8/10 (80) Cocktail* 2/5 (40) 3/3 (100) 3/3 (100) 2/2 (100) 10/13 (76.9) Pilocarpine HCl 1/2 (50) 3/3 (100) 0/1 (0) 3/6 (50) 7/12 (58.3) Phenylephrine zinc ND 1/1 (100) 0/2 (0) 2/3 (66.6) 3/6 (50) Phenylephrine HCl 2/10 (20) 2/10 (20) 5/9 (55.5) 8/10 (80) 17/39 (43.58)
Tropicamide 3/3 (100) 0/4 (0) 1/3 (33.3) ND 4/10 (40)
Homatropine HBr 1/5 (20) 1/4 (25) 1/3 (33.3) 2/5 (40) 5/17 (29.4) Atropine sulfate 0/5 (0) 0/4 (0) 2/3 (66.6) 2/5 (40) 4/17 (23.5) Cyclopentolate HCl 1/4 (25) 1/3 (33.3) 0/3 (0) ND 2/10 (20)
Artificial tears ND ND 0/1 (0) 0/1 (0) 0/2 (0)
* Cocktail was made from Atropine sulfate 1%, Phenylephrine Hcl 2.5%, Cyclopentolate HCl 1% and Tropicamide 1% (in equal proportions) in the departments.
ND: Not determined
Table 4. Contamination rates of the eye drops used in the outpatient departments with the isolated microorganisms after 1, 2, 4 or 7 days.
Number of containers in which they occurred/total (%) Contaminating organism
identified 1 Day’s use 2 Days’ use 4 Days’ use 7 Days’ use
Overall rate (%) Bacteria
Staphylococcus epidermidis 7/50 (14) 14/50 (28) 18/50 (36) 20/50 (40) 29.5
Staphylococcus aureus 1/50 (2) 3/50 (6) 5/50 (10) 8/50 (16) 8.5
Micrococcus spp 2/50 (4) 6/50 (12) 10/50 (20) 9/50 (18) 13.5
Bacillus spp 10/50 (20) 4/50 (8) 7/50 (14) 11/50 (22) 16.0
Corynebacterium spp 0/50 (0) 3/50 (6) 2/50 (4) 3/50 (6) 4.0
group A Streptococci 0/50 (0) 0/50 (0) 1/50 (2) 0/50 (0) 0.5
Streptomyces spp 1/50 (2) 0/50 (0) 0/50 (0) 0/50 (0) 0.5
Fungi
Penicillium spp 4/50 (8) 12/50 (24) 6/50 (12) 8/50 (16) 15.0
Cladosporium spp 1/50 (2) 0/50 (0) 5/50 (10) 7/50 (14) 6.5
Aspergillus niger 0/50 (0) 3/50 (6) 3/50 (6) 5/50 (10) 5.5
Aspergillus fumigatus 5/50 (10) 3/50 (6) 2/50 (4) 4/50 (8) 7.0
Aspergillus flavus 3/50 (6) 8/50 (16) 2/50 (4) 3/50 (6) 8.0
Other Aspergilli 0/50 (0) 2/50 (4) 3/50 (6) 5/50 (10) 5.0
Mucor spp 0/50 (0) 1/50 (2) 0/50 (0) 1/50 (2) 1.0
Geotricum spp 0/50 (0) 1/50 (2) 0/50 (0) 0/50 (0) 0.5
153
drops was taken into account it became evident that there were a clear-cut association between contamination rate of caps and contents with the length of time that they were used. This finding is in agreement with other reports (1, 9, 13).
Conversely, in some other reports it has been clamed that duration of use does not influence the degree of contamination.
High frequencies of microbial contamination were detected in the caps and the residual contents of the eye drops; however, the latter appeared to have higher incidences of contamination (12% vs 34% for the first day samples). While several studies have shown conflicting views (5, 7-10) with some of findings of this investigation, there are reports about lower bioburden rates (11, 12).
The results obtained for caps are comparable to the earlier reports (6, 8) in which caps of squeezed bottles have been proposed to act as potential reservoirs for microbial contamination. This contamination may make a way to the contents through the droppers.
No organism was detected on the plates of droppers in contrast to caps and residual contents.
These results are similar to those of a previous report (21) in which contents were more contaminated than tips and thus it was concluded that germ desiccation and also aspiration of contaminated tips into the content may explain higher bioburden rate of the residual contents.
Accordingly, it has been suggested that the role of preservatives is not sufficient to ensure the sterility of multi-dose eye drops during their uses and this justifies safer (single dose or filtration system) eye drop vials. Our findings on droppers, however, are not in agreement with earlier studies (1, 5, 6, 8) which have suggested the moist nozzle might serves as a reservoir for microbial contamination.
Of the eye drops tested, Tetracaine HCl, Betamethasone and the Cocktail showed higher contamination rates which were 84.4%, 80% and 76.9% respectively. The high bioburden load of the Cocktail eye drop could be attributed to the poor aseptic condition in the preparation of this product. However, since the number of some of the tested ophthalmic drops was not large enough, no conclusion could be derived on the statistical point of view.
The microbial contamination arising from the four sample groups indicated similar pattern of microorganisms. The major isolated microorganisms were either those associated with the normal skin microflora, most notably coagulase negative staphylococci and micrococci, or air-borne Gram-positive spore-bearing bacilli
and fungal spores. The only pathogenic organisms which were found were Staphylococcus aureus and group A streptococci, which are also considered as human normal flora.
Surprisingly, no Gram-negative bacteria were found in the samples. The kind of microorganisms which found in the eye drops implies that the medications have come into contact with the eyelid of patients and/or hands of nursing staffs during administration; furthermore, they may have been left without closure. The identity of contaminating organisms found in this study is consistent with those of other studies (1, 10), in which it has been demonstrated that Gram- positive cocci were the dominant contaminant of the eye drops. In another report (17) it has been shown that most of the contaminating organisms were of skin flora.
The contamination rate obtained for 1 day’s use drops was high (34%). Although most of the detected contaminants were Gram-positive organisms, in particular staphylococci and streptococci that are indigenous to conjunctiva and skin, it should be taken into account that these organisms could be harmful to patients who have disrupted epithelial barriers or those who are immunocompromised.
In conclusion, our data show that ophthalmic drops used in outpatient departments can not be utilized for more than 2 days. Yet, the contamination rate of 34% recorded for 1 day’s use medications may have a risk of infection for patients and it is necessary that the eye drops to be discarded at the end of each day. The application of multi-dose drops in outpatient departments, where a bottle is used for different patients, is potentially a source of inter-patient contamination. Ideally only single-dose eye drops should be used, but they cost more than multi- dose ones. However, application of single-dose drops is recommended for patients with eye infections. Alternatively, multi-dose bottles could be discarded after use with an infected or potentially infected patient. Meanwhile, nursing staff should be well trained to handle and administer ophthalmic preparations carefully.
ACKNOWLEDGMENT
We would like to express our gratitude to Dr. M.
Erfan of Shaheed Beheshti University of Medical Sciences for his advice on statistics. We also thank Mr. H. Jamalifar for his technical assistance.
REFERENCES
1. Geyer O, Bottone EJ, Podos SM, Schumer RA, Asbell PA. Microbial contamination of medications used to treat glaucoma. Br J Ophthalmol 1995; 79: 376-379.
Microbial contamination of ophthalmic drops 155
2. cCulloch JC. Origin and pathogenicity of Pseudomonas pyocyanea in conjucnctival sac. Arch Ophthalmol 1943; 29: 924.
3. Templeton WC, Eiferman RA, Snyder JW, Melo JC, Raff MJ. Serratia keratitis transmitted by contaminated eyedroppers. Am J Ophthalmol 1982; 93: 723-726.
4. Du Bois SK, Pinney RJ, Davison AL. Investigation of the levels of bacterial contamination in used eye drops. Pharm J 1989; 237: R39.
5. Hovding G, Sjursen H. Bacterial contamination of drops and dropper tips of in-use multidose eye drop bottles. Acta Ophthalmol 1982; 60: 213-222.
6. Coad CT, Osato MS, Wilhelmus KR. Bacterial contamination of eye drop dispensers. Am J Ophthalmol 1984; 98: 548-551.
7. Tasli H, Cosar G. Microbial contamination of eye drops. Cent Eur J Public Health 2001; 9: 162-4.
8. Schein OD, Hibberd PL, Starck T, Baker AS, Kenyon KR. Microbial contamination of in-use ocular medications. Arch Ophthalmol 1992; 110: 82-85.
9. Marion AD, Tampert MJ. Bacterial contamination of timolol in use by non-selected clinic population. Invest Ophthalmol Vis Sci 1986; 27: 36.
10. Akiba M, Yoshida L, Akiba J, Yoshida A, Tomoda Y, Tachibana M. Microbial contamination of ophthalmic solutions in examination rooms. Jpn J Clin Ophthalmol 1996; 50: 411-414.
11. Aslund B, Oslon OT, Sandell E. Studies on in-use microbial contamination of eye drops. Acta Pharm Suec 1978; 15: 389-394.
12. Stevens JD, Matheson MM. Survey of the contamination of eye drops of hospital inpatients and recommendations for the changing of current practice in eye drop dispensing. Br J Ophthalmol 1992;
76: 36-38.
13. Hugo WB, Wilson JV. Survey of in-use contamination of eye drops. J Hosp Pharm 1970; 28: 258- 260.
14. Wessels IF, Bekendam P, Calvin WS, Zimmerman GJ. Open drops in ophthalmology offices:
expiration and contamination. Ophthalmic Surg Lasers 1999; 30: 540-546.
15. Jolly S. British pharmaceutical codex. London: The Pharmaceutical Press; 1966.
16. British national formulary. London: The Pharmaceutical Press; 2001.
17. Livingstone DJ, Hanlon GW, Dyke S. Evaluation of an extended period of use for preserved eye drops in hospital practice. Br J Ophthalmol 1998; 82: 473-475.
18. Oldham GB, Andrews V. Control of microbial contamination in unpreserved eye drops. Br J Ophthalmol 1996; 80: 588-591.
19. Larsen HS. Laboratory identification of significant isolates. In: Connie RM, ed. Textbook of diagnostic microbiology. Philadelphia: W.B. Saunders Company; 2000. p. 330-399.
20. US Pharmacopeia. Validation of microbial recovery from pharmacopeial articles <1227>. In: US Pharmacopeia 25/National Formulary 20. Washington, DC: United States Pharmacopeial Convention; (2001). p. 2259-2261.
21. Raynaud C, Laveran H, Rigal D, Bonicel P. Bacterial contamination study of in-use eye drops. J Fr Ophtalmol 1997; 20: 17-24.
