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Comparative Efficacy of Topical Gatifloxacin With Ciprofloxacin, Amikacin, and Clarithromycin in the Treatment of Experimental Mycobacterium chelonae Keratitis
Joon-Young Hyon, MD;
Myung-Jin Joo, MD;
Stacey Hose;
Debasish Sinha, PhD;
James D. Dick, PhD;
Terrence P. O'Brien, MD
Arch Ophthalmol. 2004;122:1166-1169.
ABSTRACT
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Objective To determine the comparative efficacy of topical gatifloxacin with ciprofloxacin, fortified amikacin, and clarithromycin against Mycobacterium chelonae keratitis in an animal model.
Methods Experimental M chelonae keratitis was induced via intrastromal inoculation in a rabbit model. Thirty-five rabbits were randomly divided into 5 groups and each group was treated hourly for 12 hours with topical 0.9% balanced salt solution, 0.3% gatifloxacin, 0.3% ciprofloxacin hydrochloride, a combination of topical fortified amikacin sulfate (50 mg/mL) and clarithromycin (10 mg/mL), or a triple combination of topical 0.3% gatifloxacin, fortified amikacin sulfate (50 mg/mL), and clarithromycin (10 mg/mL). Antibacterial efficacy of each regimen was determined by quantitative bacteriologic analysis.
Results Treatment with 0.3% gatifloxacin or the triple combination of 0.3% gatifloxacin, topical fortified amikacin sulfate (50 mg/mL), and clarithromycin (10 mg/mL) reduced the number of mycobacterial organisms more significantly than the controls that were treated with a topical balanced salt solution (both P<.001). Therapy with 0.3% gatifloxacin was more effective than 0.3% ciprofloxacin alone (P<.001) and demonstrated synergy by enhancing the efficacy of the combination of fortified amikacin (50 mg/mL) and clarithromycin (10 mg/mL) (P<.001). Neither 0.3% ciprofloxacin nor the combination of fortified amikacin (50 mg/mL) and clarithromycin (10 mg/mL) demonstrated a significant difference in activity against mycobacteria compared with the topical balanced salt solution.
Conclusion These results suggest that topical 0.3% gatifloxacin ophthalmic solution can be a new initial treatment agent against M chelonae keratitis.
Clinical Relevance Topical gatifloxacin 0.3% may provide an initial alternative in thereapy of M chelonae keratitis.
INTRODUCTION
Infectious keratitis due to nontuberculous mycobacterial species is a rare, but devastating, complication after laser in situ keratomileusis (LASIK). Mycobacterium chelonae is reported as the most common species isolated of the nontuberculous mycobacteria causing keratitis following LASIK.1-12 Combination therapy with topical amikacin and ciprofloxacin or with amikacin and clarithromycin has been the mainstay of medical therapy.2-3,7-8 Despite aggressive and protracted treatment, the clinical outcome of medical therapy has been unsatisfactory in many cases. Ford et al13 reported that 55% of the cases of keratitis due to nontuberculous mycobacteria did not respond to medical therapy alone and ultimately required a surgical procedure.
Gatifloxacin, 1 of 8-methoxy fluoroquinolones with broad-spectrum antimicrobial activity, has been reported to be 4-fold more active than ciprofloxacin against rapidly growing mycobacteria in vitro.14 Yet, to our knowledge, no in vivo data are available for results of treatment of nontuberculous mycobacterial infections with gatifloxacin. Given its excellent in vitro activity, gatifloxacin can be considered as a potential candidate for a new therapeutic strategy against nontuberculous mycobacterial keratitis. The aim of this study was to compare the efficacy of topical gatifloxacin with ciprofloxacin, an earlier generation fluoroquinolone, and combination therapy with amikacin and clarithromycin in the rabbit model of M chelonae keratitis.
METHODS
A stock strain of M chelonae ATCC-35752 (American Type Culture Collection, Rockville, Md) was selected as the test organism. This strain demonstrated via pilot laboratory testing to induce keratitis comparable to human isolates. The suspension was diluted to 107 organisms per milliliter in phosphate buffer solution. In vitro susceptibility testing determined that the mimimum inhibitory concentration (MIC) of amikacin was 16 µg/mL (susceptible) by broth microdilution technique using National Committee for Clinical Laboratory Standards' guidelines. The reports from other separate laboratories have shown that the MIC of gatifloxacin was 0.12 µg/mL or less,14 the MIC of ciprofloxacin was 0.25 µg/mL,14 and the MIC of clarithromycin was 0.125 µg/mL15 for this carefully selected test strain.
Topical fortified amikacin (50 mg/mL) was prepared by the Johns Hopkins institutional research pharmacy from parenteral formulations according to routine procedures. Topical clarithromycin (10 mg/mL) was prepared by the research pharmacy by reconstituting an oral suspension powder with sterile water followed by dilution with artificial tears as described by Ford et al.13 Topical 0.3% ciprofloxacin (Ciloxan; Alcon Laboratories Inc, Fort Worth, Tex) was used as a commercial preparation. Topical 0.3% gatifloxacin was provided by Allergan Inc, Irvine, Calif, for the animal experiment.
Thirty-five adult male New Zealand white rabbits, weighing 3.0 to 4.0 kg, were randomly divided into 5 groups. Institutional guidelines regarding animal experimentation were followed and all animals were treated according to the Association of Vision Research in Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Anesthesia was induced with intramuscular injection of 30 mg/kg of ketamine hydrochloride (Ketaject; Phoenix Pharmaceutical Inc, St Joseph, Mo) and 5 mg/kg of xylazine hydrochloride (Xyla-ject; Phoenix Pharmaceutical Inc). Topical anesthesia was achieved with administration of 1 drop of 0.5% proparacaine hydrochloride to the rabbit eyes. Through a 30-gauge needle, a 100-µg suspension of M chelonae containing 106 organisms was inoculated into the midstroma of the right cornea. Immediately after the inoculation, 0.5 mL of dexamethasone sodium phosphate (4 mg/mL) was injected subconjunctivally to each rabbit.16
After the intrastromal inoculation, infection was allowed to proceed for 5 days before initiation of antibiotic therapy. From the fifth day after the inoculation, topical antibiotics were applied to the affected right eyes hourly for 12 hours and each group was treated with one of the following regimens: (1) 3 mg/mL of gatifloxacin; (2) 3 mg/mL of ciprofloxacin; (3) a combination of 50 mg/mL of fortified amikacin and 10 mg/mL of clarithromycin; (4) a triple combination of topical 3 mg/mL of gatifloxacin, 50 mg/mL of fortified amikacin and 10 mg/mL of clarithromycin; or (5) 0.9% balanced salt solution as a control. The rabbits were randomly allocated to each treatment group prior to intrastromal inoculation.
One hour after the final instillation of the antibiotic drops, the animals were killed with an overdose of barbiturate (Beuthanasia D-Special; Schering-Plough Animal Health Corp, Union, NJ) and uniform corneal buttons were excised with a sterile 8.5-mm trephine (Saber Medical Inc, Westchester, Pa). Corneal buttons were ground in 1-mL sterile phosphate buffer solution using a disposable tissue homogenizer (The Kendall Co, Mansfield, Mass). Then serial dilutions were prepared in sterile phosphate buffer solution. Twenty microliters from each dilution was plated on blood agar plates and incubated at 37°C with 5% carbon dioxide. Quantitative mycobacteriological analysis of viable M chelonae colonies was conducted on the sixth day of incubation.
For quantitative analysis, data were transformed to logarithmic values, and 1-way analysis of variance (ANOVA) with multiple comparisons was used to compare the efficacy of each regimen against the M chelonae keratitis. P<.05 was considered statistically significant.
RESULTS
By 4 days mycobacterial keratitis developed after inoculation in all 35 rabbits. Corneal infiltrates appeared as focal, fluffy white deposits with fine radiating projections, similar to observed and reported clinical signs in culture-proven cases of mycobacterial keratitis in humans.
The quantitative culture results are summarized in Table 1. The 0.3% gatifloxacin and triple combination therapy with 0.3% gatifloxacin, 50 mg/mL of fortified amikacin sulfate, and 10 mg/mL of clarithromycin significantly reduced the number of M chelonae compared with the control of 0.9% balanced salt solution (both P<.001). Gatifloxacin therapy demonstrated greater antimycobacterial activity than ciprofloxacin therapy (P<.001). Triple combination therapy with gatifloxacin, amikacin, and clarithromycin resulted in a more favorable result in reduction of bacterial colonies than monotherapy with 0.3% gatifloxacin, but there was no statistical significance. There was no significant difference observed between control eyes and eyes treated with ciprofloxacin or with combination therapy using 50 mg/mL of amikacin sulfate and 10 mg/mL of clarithromycin (Figure 1).
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Quantitative Mycobacterial Culture Results in Experimental Mycobacterium chelonae Keratitis*
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Results of treatment groups of 35 adult male New Zealand rabbits with Mycobacterium chelonae keratitis. Asterisks indicate P<.05; BSS, topical balanced salt solution; and CFUs, colony-forming units.
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COMMENT
Nontuberculous mycobacterial keratitis represents a persistent challenge in diagnosis and treatment because of its indolent clinical course and poor susceptibility to conventional antibacterial therapy. Most patients having nontuberculous mycobacterial keratitis report a history of antecedent trauma or prior surgery such as penetrating keratoplasty, radial keratotomy, or refractive surgery. The proper diagnosis and treatment is often delayed and the use of exogenous corticosteroid may suppress local immunity and prolong the clinical course. Biofilm of the mycobacterium may play a role in evading host defense mechanism and promoting resistance to conventional disinfection.17 Recently, there have been increasing concerns regarding mycobacteria as opportunistic pathogens associated with LASIK. Review of the ophthalmic literatures discloses 31 reported cases of post-LASIK nontuberculous mycobacterial keratitis since Reviglio et al1 first reported it in 1998. Mycobacterium chelonae was the most common pathogen identified among these cases.
The initial choice for treatment of M chelonae keratitis has been topical amikacin combined with ciprofloxacin or clarithromycin. Clarithromycin is a macrolide antibiotic with excellent in vitro activity against M chelonae,15 and topical clarithromycin has been reported to penetrate through intact epithelium.18 Amikacin also has been the drug of choice based on in vitro activity, animal studies, and clinical experiences.17 Adjunctive surgical treatment is required in many cases of M chelonae keratitis following LASIK. Among 17 cases of M chelonae keratitis following LASIK reported in the literature, 10 (59%) of 17 patients eventually underwent removal of the flap or therapeutic penetrating keratoplasty to control the infection.
In this study, gatifloxacin therapy exerted a significant antimycobacterial activity and was more active than ciprofloxacin against M chelonae keratitis. Ciprofloxacin therapy has been found to have good in vitro activity against M chelonae19 but to be less effective against M chelonae keratitis than against Mycobacterium fortuitum keratitis in vivo.20 In one series of nontuberculous mycobacterial keratitis, M chelonae isolates were generally sensitive to amikacin and clarithromycin treatment but resistant to ciprofloxacin treatment.13 Fluoroquinolnes have 2 targets on chromosomal DNA, which are DNA gyrase (type II topoisomerase) and topoisomerase IV. Microbial resistance develops when mutations occur in the target topoisomerase enzyme. The C-8 methoxy side chain of gatifloxacin may prevent bacteria from developing resistance by requiring 2 topoisomerase mutations for resistance to develop.21 Brown-Elliott et al14 reported that 97% of M chelonae isolates were susceptible or intermediately susceptible to gatifloxacin at an MIC of 4 µg/mL or less.
This study has shown that topical 0.3% gatifloxacin enhances the efficacy of topical fortified amikacin sulfate (50 mg/mL) and clarithromycin (10 mg/mL) when administered in triple combination therapy (P<.01). The efficacy of triple combination therapy with gatifloxacin, amikacin, and clarithromycin was superior to that of monotherapy with gatifloxacin in absolute colony-forming unit counts, but statistical significance was not demonstrated.
Ciprofloxacin and combination therapy with fortified amikacin and clarithromycin failed to show statistically significant antimycobacterial activity despite good in vitro activity. This observed result may be attributed to the use of corticosteroid therapy in our animal model. In 1 series of mycobacterial keratitis, 80% of the eyes with organisms that were sensitive but did not respond to topical therapy were shown to have had a corticosteroid administered.13 Corneal epithelial penetration of each antibiotic also might affect the resulting antimycobacterial activity in this study. In the animal model of this study, mycobacterial keratitis was induced by intrastromal injection of organisms that spared the intact epithelial barrier. Although topical clarithromycin has been shown to achieve therapeutic levels in corneal tissue in a rabbit model,18 topical amikacin has been reported to have poor corneal penetration.22
Although gatifloxacin and triple combination therapy with gatifloxacin, fortified amikacin, and clarithromycin have displayed significant antimycobacterial activity by reducing colony-forming unit counts in this study, cultures remained positive for M chelonae in every cornea from 35 rabbits after frequent administration of antibiotic therapy. This result advocates for a more prolonged course of medical therapy and consideration of surgical debridement to debulk organisms in the treatment of M chelonae keratitis.
To our knowledge, this is the first reported in vivo activity of gatifloxacin against M chelonae and the results confirmed the therapeutic efficacy of topical 0.3% gatifloxacin against M chelonae keratitis in an animal model. The current experiment was designed not to directly simulate clinical situations but to compare the in vivo efficacy of various treatment regimens, including different generations of fluoroquinolones, monotherapy, and combination therapy, in treating M chelonae keratitis. Thus, the experimental keratitis was treated during a short period in the early stage of the disease, which is unlikely to happen in clinical settings. Our results suggest, however, that gatifloxacin can be a valuable new initial therapeutic agent in the treatment of M chelonae keratitis. Treating clinicians should consider that multiple combination agent therapy will still be beneficial and surgical debridement may be necessary especially in post-LASIK mycobacterial keratitis to successfully eradicate these challenging pathogens.
AUTHOR INFORMATION
Correspondence: Terrence P. O'Brien, MD, Ocular Microbiology and Immunology Laboratory, Wilmer Eye Institute, 600 N Wolfe St, Baltimore, MD 21287-9121 (tobrien{at}jhmi.edu).
Submitted for publication April 30; final revision received November 17, 2003; accepted March 26, 2004.
This study was supported by the BK21 project for Medicine, Dentistry, and Pharmacy, (Dr Hyon).
From the Ocular Microbiology and Immunology Laboratory, Wilmer Eye Institute (Drs Hyon, Joo, Sinha, and O'Brien and Ms Hose), Department of Pathology (Dr Dick), The Johns Hopkins University School of Medicine, Baltimore, Md. Dr O'Brien is a nonsalaried ad hoc consultant for Alcon Laboratories Inc, Allergan Inc, Pharmacia Corp, and Santen Inc.
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