The Impact of Short-Term Topical Gatifloxacin and Moxifloxacin on Bacterial Injection After Hypodermic Needle Passage Through Human Conjunctiva

Abstract

Purpose:

To determine the bacterial contamination rate of a 27-gauge needle bore during conjunctival penetration in donor eye bank eyes and the effect of short-term use of topical 0.3% gatifloxacin and 0.5% moxifloxacin.

Methods:

One hundred consecutive human donors had 10 conjunctival penetrations per 10 syringes per eye before antibiotic placement; this was repeated 15 min after antibiotic use. Samples were cultured by expressing 0.3 mL of saline through the needle. Positive cultures were speciated.

Results:

There were 1,033 positive cultures (25.8% of all cultures); 568 (28.4%) pre-antibiotics, 249 (24.9%) after gatifloxacin (P=0.04, compared to the pre-antibiotic rate), and 216 (21.6%) after moxifloxacin (P<0.001). The most common organism was Staphylococcus epidermidis [334 positive cultures (8.4%)]. No antibiotic effect was seen on this or other organisms except S. aureus [4.6% pre-antibiotic, 2.8% after gatifloxacin (P=0.02), and 1.8% after moxifloxacin (P<0.001)] and other Staphylococcus species [5.3% pre-antibiotic, 3.6% after gatifloxacin (P=0.04), and 3.2% after moxifloxacin (P=0.01)].

Conclusions:

Transconjunctival penetration often results in needle bore contamination; bacteria are included in an injected solution. Fifteen minutes of exposure to 2 topical antibiotics had a minimal effect on bacterial contamination and no significant effect on many common pathogens.

Introduction

From intraocular triamcinolone to vascular endothelial growth factor (VEGF) inhibitors, the once uncommon procedure of intraocular injection has become exceedingly common in many retinal practices. With new indications for VEGF inhibitors rapidly multiplying, intraocular injections will continue to be commonplace in ophthalmology for the foreseeable future.

Although relatively uncommon, infectious endophthalmitis as a result of an intraocular procedure is a highly feared complication. The cumulative incidence of this complication in 24 published studies representing 176,799 intraocular injections was 0.05% (93 infections).^1–24 Many of these studies did report an incidence greater than 0.1%; however, this means that while the single injection risk is very small, a cumulative risk at 8 injections per year for 10 years would be a not so insignificant 4%. Obviously, any reasonable way to minimize the risk of infection from intraocular injections would be widely adopted.

Bacteria from an intraocular injection can be introduced into the vitreous body in only a few ways. The most obvious route is by contamination of the hypodermic needle as it penetrates the conjunctiva. Any part of the needle that penetrates both the conjunctiva and the sclera could contaminate the vitreous body. However, the cutting face of the needle projects very little surface area, and any bacteria that might adhere would likely be wiped off in penetrating the sclera. Furthermore, if bacteria have continued to adhere after passing through the sclera, then the same tendency is likely while the needle is in the vitreous. The same would be the case for adherence to the outside of the needle. However, any bacteria that falls into the bore of the needle would be protected and pass with the injected drug into the vitreous cavity. Most likely, this is an avenue of risk for causing intravitreal bacterial contamination.

Currently, infectious prophylaxis generally centers around 2 approaches: (1) the use of antibiotics and antiseptics, and (2) injection approaches that minimize any external exposure to the needle track. Concepts such as not lining up the conjunctival and scleral needle tracks and angling the injection through the sclera have generally been adopted. This study focuses on the impact of short-term topical fourth generation fluoroquinolones on the risk of bacterial contamination of the needle bore secondary to conjunctival penetration.

Methods

The University of Utah Institutional Review Board exempted this study from IRB approval. The study was conducted under an agreement with the Utah Lions Eye Bank. Forty tuberculin syringes with 27-gauge 1/2′′ needles were filled with 0.3 mL of the balanced salt solution (BSS; Alcon Surgical, Ft. Worth, TX). Corneas from 100 consecutive human donors were harvested soon after death, while the eyes remained in the cadaver head. As the first step, 10 syringes for each eye were used to penetrate the medial or temporal conjunctiva 10 times each, taking care to avoid previous areas of penetration and to avoid the lid margins; then, the needle was recapped. The 2 eyes were then randomized either to 0.5% moxifloxacin without preservative or to 0.3% gatifloxacin. Five drops of the antibiotic solution were then placed on the eye, ensuring that the fornices were covered with antibiotics. After 15 min, cellulose sponges were used to remove all visible antibiotic solution from the surface of the eye, and the remaining 20 syringes were used to penetrate the medial or temporal conjunctiva in the same fashion (10 conjunctival penetrations per syringe and 10 syringes per eye).

Upon return to the eye bank, 40 blood agar plates were used to plate the contents of the syringe. 0.3 mL of BSS in the syringe was slowly ejected through the attached needle in a circular fashion onto the surface of the agar plate. The plates were placed in an incubator at 37°C, with low partial pressure carbon dioxide added to the incubator chamber. The plates were checked daily for bacteria growth. If no growth was noted by day 14 of incubation, the plate was recorded as having no growth. Any growth was so recorded and transferred to a holding media. Samples were transferred to blood agar and incubated overnight at 37°C. Individual colonies were isolated, Gram-stained, and bacteria were speciated using the API Bacterial Identification Test (Gram-positive organisms; BioMérieux, Durham, NC) or BBL™ Enterotube™ II (Gram-negative organisms; Becton, Dickinson and Company, Franklin Lakes, NJ) systems.

As a pilot study using the same technique without any antibiotic, 6 cadaver eyes had the conjunctiva penetrated in the same manner (10 needles and 100 penetrations per eye) and the results cultured in the same fashion.

For statistical analysis, we compared the positive culture rate before and after antibiotic use, as well as the positive culture rate between the 2 antibiotics, by a Chi-square analysis. Significance was set at P<0.05.

Results

We obtained 4,000 samples, 2,000 before antibiotic instillation (representing 20,000 penetrations of the conjunctiva) and 1,000 in each of the 2 randomized groups after instillation. There were positive cultures in 1,033 of the 4,000 samples (25.8%), 282 of 1,000 cultures for the right eye (28.2%) and 286 of 1,000 cultures in the left eye (28.6%), showing the effect was likely consistent, for a final positive pre-antibiotic total of 568 (28.4%). These results and the impact of the antibiotics are shown in Table 1. Both antibiotics had a small, but significant decrease in positive cultures and were not significantly different from each other.

Table 1.

Positive Bacterial Cultures After Passing a 27-Gauge Needle Through Human Cadaver Conjunctiva Before and After 5 Drops of a Topical Antibiotic Solution Were Placed on the Ocular and Conjunctival Surface for 15 Min

	Pre-antibiotic	After 0.3% gatifloxacin	After 0.5% moxifloxacin
Number of samples	2,000	1,000	1,000
Positive cultures	568 (28.4%)^a,b	249 (24.9%)^a	216 (21.6%)^b
Staphylococcus epidermidis	168 (8.4%)	86 (8.6%)	80 (8.0%)
S. aureus	92 (4.6%)^c,d	28 (2.8%)^c	18 (1.8%)^d
Other staphylococcal species	106 (5.3%)^e,f	36 (3.6%)^e	32 (3.2%)^e
Gram-negative organisms	64 (3.2%)	19 (1.9%)	22 (2.2%)
Micrococcus	48 (2.4%)	17 (1.7%)	15 (1.5%)

Each sample represents 10 conjunctival penetrations.

P=0.04.

P<0.001.

P=0.02.

P<0.001.

P=0.04.

P=0.01.

Speciation was possible in 869 of the 1,033 plates with a positive culture. One hundred thirty-one cases showed 2 different organisms, 4 showed 3 different organisms, and the rest showed 1 organism. Of the 170 plates not speciated, 48 were typified as Gram-positive or Gram-negative, and were identified as a rod or a coccus. Table 2 lists the most common of the 30 species identified. Table 1 shows the effect of the antibiotics on common pathogens with the most common pathogen causing endophthalmitis, Staphylococcus epidermidis, not significantly decreased by the use of either antibiotic.

Table 2.

Organisms Obtained from 27-Gauge Needle Sampling Penetrations of Cadaveric Human Conjunctiva

Species	Number of positive cultures
S. epidermidis	334
Other staphylococcal species (not S. aureus or S. epidermidis)	174
S. aureus	138
Gram-negative organisms	105
Micrococcus species	80
Fungal species	80
Gram-positive cocci not further speciated	8
Gram-positive rods not further speciated	5
Streptococcus species	3

One thousand thirty-three positive cultures, of which 131 showed 2 different species and 4 showed 3 species. Those cultures that could not be speciated are not listed here.

Table 3 shows all eyes that had a positive culture. As can be seen, there is a strong concordance between pre-antibiotic and post-antibiotic positive cultures; however, there were quite a few eyes that were positive after antibiotic use and negative before the antibiotic was placed. This is the nature of the random sampling of the conjunctival tissue that needle penetration would represent.

Table 3.

Culture Rates of Eyes That Were Positive Post-Antibiotic and Positive Pre-Antibiotic

Number of eyes with positive cultures
Post-moxifloxacin	78/100
Post-gatifloxacin	77/100
Of eyes with positive cultures post-antibiotic, number of eyes with positive cultures pre-antibiotic
Moxifloxacin	67/78	86%
Gatifloxacin	68/77	88%

This difference between pre- and post-antibiotic culture rates is likely due to the random nature of conjunctival sampling with the needle.

The pilot study looking at the impact of decreasing the needle gauge to 30 had 12 positive cultures out of 60 cultures for a culture-positive rate of 20%, which is not statistically different than our results with the 27-gauge needle.

Discussion

Although the incidence of endophthalmitis after intraocular injection is very small,^1–24 with the increasing therapeutic use of this approach and the increasing number of injections per patient, the cumulative risk is an important public health issue. A meta-analysis published in 2004 placed the incidence at 0.2% after intravitreal injections, so the most recent average from these 24 articles of 0.05% may represent progress in decreasing this feared complication.²⁵ Nonetheless, it is important to consider how the bacteria might gain access to the eye. The small needle track may create exposure to the contaminated conjunctiva or even the tear film if there is a vitreous strand protruding from the scleral incision; however, if the conjunctival and scleral penetrations are discontinuous and the scleral penetration is angled so that the intraocular pressure will collapse this very small scleral opening, this would seem unlikely. This study suggests bacteria that are captured in the bore of the needle as it passes the conjunctiva, and then incorporated as part of the injection volume, as another possible route for intraocular contamination. We know that the vitreous humor has very little ability to overcome a bacterial load,²⁶ so that direct injection of bacteria into the vitreous body is a scenario likely to cause infection at least some of the time.

We have been able to show that the presence of infectious organisms in the bore of the needle was a common event in our scenario. Our pre-antibiotic-positive culture rate was 28.4%, representing 10 conjunctival penetrations. Thus, the lowest rate of bacteria being trapped in the bore of the needle during any one conjunctival penetration would be about 2.8% per injection if only 1 penetration in the 10 were to pick up bacteria. The exact number is difficult to extrapolate because bacteria could have been picked up more than once per the 10 penetrations per sample. However, we believe that 2%–3% is a reliable estimate. Our scenario cannot duplicate the present practice of intraocular injection in patients; however, it would seem difficult to consider that transecting the conjunctiva with a hypodermic needle would not pick up bacteria in the bore of the needle at least some of the time.

In a recent study, swabs were used to culture the conjunctival surface before intravitreal injections in patients. One hundred thirty-six samples were taken from 90 eyes of 85 patients, and 65 positive cultures resulted. Cultures showed 71 positive strains, of which 59 were coagulase-negative staphylococci (CNS; 83%).²⁷ In a second, similar study, CNS represented 65% of positive conjunctival cultures before intravitreal injections, of which 47% was S. epidermidis, the majority of which were multidrug resistant.²⁸ A meta-analysis of endophthalmitis after intravitreal injection showed 65% of culture-positive cases were CNS.²⁹ Results of both studies are greater than the 32% of cultures that were S. epidermidis in our study. Possibly, surface swab cultures do not accurately reflect what a needle core biopsy of the conjunctiva represents due to the folds and crypts of the conjunctival surface. Because it is the needle core biopsy of the microorganisms associated with the conjunctiva that will be eventually injected into the vitreous, our results may be of a greater clinical relevance.

The primary aim of our study was to assess the impact of topical antibiotics 15 min after their application. The antibiotic was allowed to pool over the conjunctiva for the entire time without a decrease in concentration due to gravity, tearing, and blinking, as would occur in a living patient. While significant, both fourth-generation fluoroquinolones had only a small effect in decreasing the odds that the needle bore would pick up bacteria. Furthermore, one should consider that the bore of the needle, besides picking up bacteria, was likely to pick up antibiotic despite the conjunctival surface drying that occurred. This antibiotic effect in the needle bore would impact bacteria that were present until culturing occurred, which often took place an hour after the experiment. This would not be an issue with immediate injection into the vitreous body, where the small antibiotic effect would be immediately and drastically diluted. In an actual clinical scenario, this antibiotic effect would likely be even less than the small effect noted here. We therefore conclude that frequently used topical fluoroquinolones if used shortly before an intraocular injection have little impact on the incidence of injecting bacteria into the eye.

Of the bacteria recovered, the most important causative organisms for endophthalmitis are S. epidermidis (8.4% of all cultures), S. aureus (3.45% of all cultures), other staphylococcal species (4.35% of all cultures), Micrococcus species (2.0% of all cultures), Streptococcus species (only 3 speciated), and Gram-negative organisms (2.6% of all cultures). Only S. aureus and other non-S. epidermidis and non-S. aureus species showed a significant decrease in numbers after the antibiotic was placed. Furthermore, a large number of positive cultures (170) could not be speciated, and our culture method (blood agar with 5% CO₂ in the incubator) would not be effective for many fragile organisms. Therefore, the scope of the problem is likely much larger than we were able to document. Nonetheless, we believe that our results capture most of the common pathogens usually encountered in cases of endophthalmitis.

This study also has relevance to eye banking. Relatively short-term use of topical antibiotics is commonly used with other antibacterials in an attempt to sterilize the globe. Our results demonstrated that 15 min of antibiotics had only a small effect on decreasing the conjunctival bacterial load, and no significant effect on many common pathogens. The antibiotic effect on sterilizing the corneal surface for tissue banking also deserves additional studies.

Topical povidone-iodine is also commonly used both for eye banking and for intraocular injection prophylaxis. The impact of such antisepsis on what is essentially a sampling of the conjunctiva by the bore of the needle is the subject of a similar study of povidone-iodine, which is ongoing in our laboratory. Preliminary results have not documented substantial efficacy of topical povidone-iodine; consequently, povidone-iodine may not be the panacea for antisepsis that so many believe it to be. In another study, we observed that gatifloxacin 0.3% (Zymar), which contains the preservative benzalkonium chloride, had a greater antibacterial activity than did gatifloxacin alone in resistant S. aureus, thus illustrating potential advantages of this preservative.³⁰ Other studies also have demonstrated improved efficacy of ophthalmic solutions containing preservative over preservative-free solutions.^31–33 Despite theoretical differences, showing this impact clinically has not been documented and our results in this study, if anything, favored the effect of unpreserved moxifloxacin, although the differences were not statistically significant.

If the major risk from endophthalmitis does come from the needle bore's picking up bacteria as it passes through the conjunctiva, then we can reasonably conjecture what might and might not be efficacious from a prophylaxis standpoint. Decreasing the bacterial load would likely be more effective with several days of antibiotics before surgery, as has been shown from culture studies before cataract surgery.^34,35 However, the Moss study did not show that the antibiotic effect was significantly additive to the anti-bacterial effect of topical povidone-iodine alone. In fact, Scott and Flynn, in an editorial in 2007, questioned whether antibiotics had a role at all in endophthalmitis prophylaxis associated with intraocular injections.³⁶ Therefore, povidone-iodine antisepsis alone may be all that is necessary to decrease the bacterial load of the conjunctiva. However, the subject has not been thoroughly studied to date. In the Moss study, the numbers were quite small, with inadequate statistical power to detect a difference. Using our model to study needle core conjunctival biopsy after topical povidone-iodine should help to answer this question.

Decreasing the bore size, perhaps to a 31-gauge needle as is often used, may decrease the likelihood of picking up bacteria; however, we did try 30-gauge needles with 60 penetrations in 6 eye-bank eyes as a small pilot study. We had 12 positive cultures (20%), so decreasing needle bore may not have much impact on decreasing needle bore contamination. We had picked 27-gauge needles as the upper end of this risk because they are commonly used for the intraocular injection of triamcinolone. Because penetration of any topical antibiotic into the vitreous is minimal and does not reach therapeutic levels with typical delivery frequencies,³⁷ the use of topical antibiotics after the injection probably does not make sense, unless vitreous prolapses from the resulting needle penetration site. How often this might occur is not presently known; however, this risk should also be directly related to the size of the needle, so very small needles and an angled injection should minimize this risk. Use of postinjection oral antibiotics that do get into the vitreous at therapeutic levels may be a more sensible approach; however, the cost and risk of systemic complications, if a therapeutic course is truly implemented, are daunting barriers to consider. Until additional studies are conducted, the mainstays of prophylaxis are (questionably) pre-operative antibiotics (preferably for more than a few minutes before injection), topical antisepsis, and the smallest possible bore needle combined with appropriate injection techniques (angled injection and making the conjunctival and scleral injection sites discontinuous).

Limitations of our study include the use of human cadaver eyes in which the bacterial load will increase after death, and the unnatural concentration of antibiotics due to loss of the effects of gravity, blinking, and tearing. It also is possible that the act of washing the eye surface before use of cellulose sponges to remove the visible solution could have decreased the bacterial counts, irrespective of the type of fluid used. Furthermore, we did not control for the temperature of the cadaver eyes, which could have had an impact on our results. Recording surface temperatures of cadaver eyes, and correlating this variable with antimicrobial effects, might prove to be an interesting addition to future studies. Repeating these results in patients would not be ethically possible; and using endophthalmitis-positive cultures in any human randomized study would require millions of patients to be enrolled to get bacteriological results equivalent to those reported here. While the differences in conjunctival bacterial loads between human cadavers within hours of death and patients are not known, our main focus was the impact of bacterial prophylaxis on the local conjunctival flora. Knowing that antibiotics pooled on the conjunctiva for 15 min did little to decrease the bacterial load and had no effect on common pathogens strongly suggests that short-term preinjection topical antibiotic prophylaxis is relatively ineffective.

In conclusion, we showed that bacteria were picked up in the bore of a 27-gauge needle, after it passed through human cadaver conjunctiva, ∼2.8% of the time per each pass through the conjunctiva, and that they are included in the infusate after injection. We surmise that this is a potential major risk factor for endophthalmitis after intraocular injections, and that 15 min of topical soaking with either 0.3% gatifloxacin or 0.5% moxifloxacin has only a small effect in decreasing this bacterial count. Furthermore, these antibiotics had no significant effect on common pathogens such as S. epidermidis, Gram-negative bacteria, and Micrococcus. We recommend that the goal of prophylaxis for injection-associated endophthalmitis be to decrease the conjunctival bacterial load before surgery, although the best way to do this remains controversial. Topical antisepsis with topical povidone-iodine is probably more important than the use of topical antibiotics; however, this deserves more clinical study. Using a needle bore that is as small as possible, and a technique in which the conjunctival and scleral penetration are discontinuous and angled through the sclera, also makes sense.

Footnotes

Acknowledgments

The Utah Lions Eye Bank at the University of Utah provided assistance with implementation of this study. Susan Schulman provided writing assistance with the article. Matthew Huff provided technical assistance. This study was supported, in part, by an unrestricted grant from Research to Prevent Blindness, Inc., New York, NY, and, in part, by an unrestricted grant from Allergan, Inc., Irvine, CA, to the Department of Ophthalmology and Visual Sciences, University of Utah. This study was also supported, in part, by unrestricted grants from Allergan, Inc., to the Department of Ophthalmology, University of Oklahoma Health Sciences Center and from Research to Prevent Blindness to the Dean A. McGee Eye Institute.

Proprietary Interest

The authors do not indicate any proprietary interest.

Author Disclosure Statement

No competing financial interests exist.

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