Graefes Arch Clin Exp Ophthalmol (2013) 251:2111–2117 DOI 10.1007/s00417-013-2371-y
BASIC SCIENCE
Evaluation of the in vitro activity of commercially available moxifloxacin and voriconazole eye-drops against clinical strains of Acanthamoeba C. M. Martín-Navarro & A. López-Arencibia & F. Arnalich-Montiel & B. Valladares & J. E. Piñero & J. Lorenzo-Morales
Received: 10 January 2013 / Revised: 8 April 2013 / Accepted: 25 April 2013 / Published online: 19 May 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Purpose Acanthamoeba is an opportunistic pathogen which is the causal agent of a sight-threatening ulceration of the cornea known as “Acanthamoeba keratitis” (AK) and, more rarely, an infection of the central nervous system called “granulomatous amoebic encephalitis” (GAE). The symptoms of AK are non-specific, and so it can be misdiagnosed as a viral, bacterial, or fungal keratitis. Furthermore, current therapeutic measures against AK are arduous, and show limited efficacy against the cyst stage of Acanthamoeba. Moxifloxacin, a fourth generation fluoroquinolone, has been used with other drugs to treat GAE, but its efficacy as a treatment for AK is not known. Voriconazole has been used to treat AK; however, its cysticidal efficacy is not known. Both drugs are commercially available as eye-drops. The aim of this study was to evaluate the in-vitro activity of these eye-drops against Acanthamoeba compared to two reference drugs (chlorhexidine and amphotericin B) which are currently used to treat AK and GAE. Methods The sensitivity of two clinical and one type strain of Acanthamoeba to the commercial concentrations of the four drugs was evaluated with a colorimetric assay. Mature
cysts were incubated with voriconazole to determine their sensitivity to this drug. The effects on cell proliferation and cell toxicity were determined using standard procedures with commercial kits. Results The four compounds were active against the Acanthamoeba strains in this study. Although it prevented encystation, moxifloxacin’s amoebicidal activity was low. Voriconazole activity was greater than that of the other drugs, even at a concentration lower than in commercial eye drops. It was effective against cysts and decreased cell proliferation, with low cellular cytotoxicity. Conclusion Voriconazole could be used against AK as a first-line treatment or in combination. Moxifloxacin is an interesting adjuvant to consider as it is effectively prevents encystation of the amoeba which often complicates infection resolution. In addition, moxifloxacin is effective in preventing secondary bacterial infections.
C. M. Martín-Navarro : A. López-Arencibia : B. Valladares : J. E. Piñero : J. Lorenzo-Morales University Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, Avda Astrofísico Fco. Sánchez, S/N, 38203 La Laguna, Tenerife, Canary Islands, Spain
Introduction
C. M. Martín-Navarro (*) Centre for Integrative Physiology, University of Edinburgh, Scotland, UK e-mail:
[email protected] F. Arnalich-Montiel Cornea Unit, Department of Ophthalmology, Ramón y Cajal Hospital, Madrid, Spain
Keywords Acanthamoeba . Acanthamoeba keratitis . Therapy . Moxifloxacin . Voriconazole
Acanthamoeba, a free-living amoeba with wide distribution, may cause opportunistic infections in humans. Pathogenic strains of Acanthamoeba are the causative agents of a sightthreatening ulceration of the cornea known as Acanthamoeba keratitis (AK) and also amoebic encephalitis (granulomatous amoebic encephalitis, GAE). Acanthamoeba has been classified in 17 different genotypes (T1–T17) based on rRNA gene sequencing [1–7]. To date, studies have shown that 90 % of Acanthamoeba isolates related to infections belong to the T4 genotype [8]. In recent years, the incidence of AK has shown a remarkable increase, which is directly related to a higher
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number of contact lens wearers [9, 10]. The most important risk factors for AK in developed countries are ineffective lens disinfection systems, the use of home-made saline solution and contaminated lens storage cases [8, 11–14]. Moreover, AK symptoms are not specific, and it is often misdiagnosed as a viral, fungal, or bacterial keratitis. To date, no single agent has been described as being an effective treatment against AK. Although Acanthamoeba trophozoites are sensitive to most available chemotherapeutic agents (antibiotics, antiseptics, antifungals, etc.), persistent infection is related to the presence of the Acanthamoeba cyst, which is the resistant stage of this pathogen [15–19]. Diamidines (propamidine and hexamidine) and biguanides [clorhexidine and polyhexamethylene biguanide (PHMB)] are currently recognized as being the most effective antiamoebic agents. The in-vitro cycsticidal activity of these drugs has been studied, and a series of clinical cases support their therapeutic efficacy [8, 10, 19–22]. However, about 5 % of patients with AK have persistent inflammation due to viable Acanthamoeba in the cornea, even after prolonged treatment with these biguanides and/or diamidines [23]. There is a clear need for new therapeutic agents against Acanthamoeba. Most of the current approaches are focusing on targeting key proteins mainly related to cellular viability and amoebic pathogenesis, but recently, moxifloxacin (a fourth-generation synthetic fluoroquinolone) has been successfully used in combination with other compounds for the treatment of GAE [24]. Another active compound that has been successfully used in the clinical practice is voriconazole, even in cases of AK caused by resistant strains [25, 26]. However, to the best of our knowledge the cysticidal activity of voriconazole has not been reported before. Furthermore, these drugs are of high interest, since they are both commercially available as eye-drops. The aim of this study was to evaluate the in-vitro activity of these eye-drops against Acanthamoeba and to compare their activity with two reference drugs (chlorhexidine and amphotericin B) which are being currently used in cases of AK and GAE.
Methods Acanthamoeba strains A type strain, Acanthamoeba castellanii Neff (ATCC 30010, genotype T4) and two clinical strains (CLC-16, genotype T3 and CLC-51, genotype T1) isolated from contact lens cases and characterized as highly pathogenic in a previous study [14, 27] were used in this study. These strains were axenically grown in PYG medium [0.75 % (w/v) proteose peptone, 0.75 % (w/v) yeast extract and 1.5 % (w/v) glucose] containing 40 μg/ml gentamicin
Graefes Arch Clin Exp Ophthalmol (2013) 251:2111–2117
(Biochrom AG, Cultek, Granollers, Barcelona, Spain) at room temperature. Chemicals Four antimicrobial preparations available for topical use against Acanthamoeba keratitis were selected for analysis. The preparations included a biguanide (clorhexidine), a fourth-generation quinolone (moxifloxacin), and two antifungal agents (voriconazole and amphotericin B). Moxifloxacin topical solution (0.5 %, 5 mg/ml) was purchased from Alcon Laboratories (Barcelona, Spain). The other three drugs were prepared at the commercially-available concentrations. Voriconazole (VFEND®) was provided by Roerig/Pfizer Inc. (New York, USA) and reconstituted in 50 % of WFI (water for injection) and 50 % BSS (balanced salt solution) according to the manufacturer's specifications, reaching a final concentration of 1 % (10 mg/ml). Clorhexidine digluconate was purchased from Alfa Aesar (Barcelona, Spain) and amphotericin B was provided by Sigma-Aldrich Chemistry Ltd (Madrid, Spain). Both drugs were diluted in bi-distilled sterile water to reach a final concentration of 0.1 % (1 mg/ml). The working concentrations mentioned above are the concentrations of these active compounds presented in the commercially available eye-drops of each of these drugs. Amoebicidal activity assays The anti-Acanthamoeba (anti-trophozoite) activity of each agent was determined using the alamarBlue Assay Reagent® as previously described [14, 28, 29]. The assay measures the innate cellular metabolic activity, as it induces a redox reaction of the alamarBlue dye. The redox process causes a change of colour of the dye, and works as a measurable indicator of the amount of viable cells that are present in a sample. Testing was performed using a 96-well, flat-bottomed microtiter plate assay (Nunc, Thermo Fisher Scientific Inc., Madrid, Spain). Briefly, 50 μl of each Acanthamoeba strain (104 cells/ml) were seeded in duplicate in the 96-well microtiter plates and were allowed to adhere for 15 min. After that, 50 μl of two-fold serial dilutions in PYG medium, of the different compounds were added to each well. Finally the alamarBlue® (Biosource, Europe, Nivelles, Belgium) was placed into each well at an amount equal to 10 % of the final volume which was 100 μl. Test plates containing the alamarBlue® were then incubated for 120 h at 28 °C with slight agitation. The tested concentrations varied between products, but included the concentrations of these drugs presented in the commercially available eye-drops. Thus the range of tested concentrations was as follows: 5 mg/ml to 0.039 mg/ml for moxifloxacin, 10 mg/ml to 0.078 mg/ml for voriconazole, and from 100 μg/ml to 0.39 μg/ml for chlorhexidine and amphotericin B.
Graefes Arch Clin Exp Ophthalmol (2013) 251:2111–2117
The plates were subsequently analysed between 72 and 120 h, with a Microplate Reader Model 680 (Biorad, Hercules, CA, USA), using a test wavelength of 570 nm and a reference wavelength of 630 nm. Percentages of amoebic growth inhibition, 50 % inhibitory concentrations (IC50), and 90 % inhibitory concentrations (IC90) for each agent were calculated by linear regression analysis, with 95 % confidence limits. All experiments were performed three times each in duplicate, and the mean values were also calculated. A paired two-tailed t-test was used for analysis of the data. Values of p
