In Vitro Comparison of Efficacy of Natamycin and Silver Nitrate against Ocular Fungi ACCEPTED

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1 AAC Accepts, published online ahead of print on 12 January 2009 Antimicrob. Agents Chemother. doi: /aac Copyright 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved In Vitro Comparison of Efficacy of Natamycin and Silver Nitrate against Ocular Fungi Yan Xu 1*, Guangren Pang 1, Chuanwen Gao 1, Dongqing Zhao 1, Lutan Zhou 1, Shengtao Sun 1, Bingliang Wang 2 Henan Institute of Ophthalmology, Department of Ophthalmology, Henan Provincial People s Hospital 1, and Anyang Eye Hospital 2, China * Corresponding author. Mailing address: Department of Ocular Pharmacology, Henan Institute of Ophthalmology, 7 Weiwu Road, Zhengzhou , China. Phone: , Fax: xuyan990301@yahoo.com.cn Abstract The in vitro activity of the silver nitrate was assessed in comparison with that of natamycin against 128 corneal Fusarium isolates and 90 corneal Aspergillus isolates. MIC 90 values of silver nitrate were 2µg/ml for Fusarium spp., 1µg/ml for Aspergillus spp. MIC 90 values of natamycin were 8µg/ml for Fusarium spp., 32µg/ml for Aspergillus spp. Silver nitrate exhibited potent antifungal activity against ocular fungi in vitro. The blindness caused by keratomycosis is a serious health problem in the developing world. Keratomycosis has become increasingly prevalent in corneal diseases that are responsible for the vision loss in China (17,24,26). Clinical studies indicate that keratomycosis constitutes about 46.7% to 61.9% of all cases of suppurative keratitis inpatients, and 35.1% of all cases of infectious keratitis underwent penetrating keratoplasty in some regions in China (3,24,26). Filamentous fungi, mainly Fusarium spp. 1

2 or Aspergillus spp., are the most frequently isolated fungi in patients with keratomycosis and the most common ocular pathogenic fungi in China. To date, keratomycosis still remains a therapeutic challenge for the ophthalmologist, as the clinical management has been limited due to the relative unavailability of effective antifungal agents. Corneal lesions fail to resolve in many of patients who receive antifungal treatment and some patients get marked loss of vision and eventually perforation of the cornea, ultimately require penetrating keratoplasty; or even enucleation or evisceration (15,17,19,24). Therefore, it is very important and urgent to develop new broad-spectrum antifungal agents that are active against a wide variety of ocular fungal pathogens, perhaps available as antifungal eye drops that combat this vision-threatening infection in the ophthalmologic clinic. The use of metallic silver as an antimicrobial agent has long been recognized (6,8). Silver nitrate had been used since the 19th century to prevent ophthalmia neonatorum and treat infections and burns (2, 6, 13). Silver compounds possess the advantage of having broad antimicrobial activities against bacteria, viruses and fungi, with minimal development of microbial resistance (13). However, the efficacy of silver against ocular pathogenic fungi in vitro has not been evaluated so far. The present study was to determine the antifungal activity of silver nitrate against ocular pathogenic fungi in vitro, and compare its effects with those of natamycin, the first and until recently the only topical ophthalmic antifungal compound approved in the United States (12). 2

3 Fusarium spp. isolates and 90 Aspergillus spp. isolates from patients with keratomycosis from Henan Institute of Ophthalmology in Zhengzhou, China were investigated. These isolates were identified based on morphology by standard methods (16,21-23). Candida parapsilosis ATCC22019 was used as quality control. The antifungal agents tested in this study were silver nitrate (Fourth Air Pharmaceutical Factory, Wuhan, China, minimum 99.8%) and natamycin (Yinxiang Biotechnology Co. LTD, Zhejing, China, minimum 95%). The both were dissolved in 100% dimethyl sulfoxide, respectively. The stock solutions were prepared at concentration of 1600µg/ml and then were stored at -65 C until tested. All dilutions were made in RPMI 1640 medium (with L-glutamine, without sodium bicarbonate, GIBCO BRL, Grand Island, N.Y., USA) buffered to ph 7.0 with M morpholinepropanesulfonic acid (MOPS, Serva, Feinbochemica GmbH, & Co., Germany) and 10 M NaOH. Final concentrations for both drugs ranged from to 16 µg/ml. A broth microdilution method was used following the Clinical and Laboratory Standards Institute (CLSI) M38-A document (11) which describes a standard method for testing the susceptibility of conidium-forming filamentous fungi that cause invasive fungal infections, including Aspergillus species, Fusarium species etc. to antifungal agents. Inocula were prepared in accordance with the CLSI M38-A document. The final inoculum was 0.4 x 10 4 to 5 x 10 4 CFU/ml. 3

4 Following incubation at 35 C for 48 h, MIC was determined according to the CLSI M38-A document. For both agents tested, MIC was defined as the lowest drug concentration that prevented any discernible growth. MIC range, Mode, MIC 50 and MIC 90 were provided for the isolates with the statistical SPSS package (version 13.0). For calculation, the high off-scale MICs were converted to the next concentration up. The in vitro activities of silver nitrate and natamycin against the Fusarium spp. and Aspergillus spp. were summarized in Table 1. Both MIC 50 and MIC 90 of silver nitrate were 2µg/ml for Fusarium spp, 1µg/ml for Aspergillus spp. respectively. Both MIC 50 and MIC 90 of natamycin were 4µg/ml and 8µg/ml for Fusarium spp, 32µg/ml for Aspergillus spp. respectively. Our results show that MIC 90 of natamycin were 8µg/ml for Fusarium spp and 32µg/ml for Aspergillus spp. Our findings in this study suggest that natamycin is ineffective against Aspergillus spp. and mildly or moderately effective against Fusarium spp. in vitro. The results are in agreement with some treatment failure using natamycin for patients with keratomycosis (4,9,18). The results of this study suggest that silver nitrate may be superior to natamycin in potency against corneal Fusarium and Aspergillus isolates in vitro. Particularly, compared with the MIC 90 values between the both, activity of silver nitrate against Aspergillus spp. is 32 times greater than that of natamycin, and 4 times higher than 4

5 natamycin when against Fusarium spp. In an earlier study, Wan introduced the application of 3% silver nitrate ophthalmic solution to the eyes of clinical keratomycosis. A higher success rate was achieved with silver nitrate (78%) than without silver nitrate (46%) treatment (20). In a recent study, Zhang reported that in keratomycosis, 92% of 38 patients showed a favorable response to subconjunctival injection of 2.5mg amphotericin B once daily, or on alternate days once and an adjuvant 0.5% silver nitrate drops on alternate days once. The study showed that silver nitrate may have an adjunctive role in the management of keratomycosis (25). Our research result that silver nitrate exhibits potent antifungal activity against corneal Fusarium and Aspergillus isolates in vitro is in accordance with some good outcomes with silver nitrate for patients with keratomycosis. Wan and Zhang reported that no significant ocular or systemic adverse effects were observed during the study periods (20,25). During the 20th century, silver nitrate eye drops instillation to newborns had been the best prophylaxis against ophthalmia neonatorum caused by Neisseria gonorrhea (5, 7, 14). To date, in the developing world and some developed countries, silver nitrate has still been used for prophylaxis in the neonatal period because of its effect against gonococcus (1,10). Chemical conjunctivitis is much more commonly seen with silver nitrate eye prophylaxis than with other medications include erythromycin, tetracycline, gentamicin and 2.5% povidone-iodine ophthalmic solution (1,5,10,14). But silver nitrate eye prophylaxis caused no sustained deleterious effects and even provided some benefit to infants born to women without Neisseria gonorrhoeae(5,10). 5

6 In conclusion, in vitro susceptibility data suggest that silver nitrate may be effective against corneal Fusarium and Aspergillus isolates and a prospective evaluation of efficacy and safety would be required to further develop its clinical applications. REFERENCES 1. Assadian, O., A. Assadian, C. Aspöck, D. Hahn, and W. Koller Prophylaxis of ophthalmia neonatorum--a nationwide survey of the current practice in Austria. Wien Klin Wochenschr.114: Crede, C. S. R Die verhutung der augenentzundung der neugeborenen. Archiv Gynaekol. 17: Deng, X. G., X. F. Lu, and G. R. Pang Analysis of the cause and pathogen of the pyogenic corneal ulcer in 312 cases. Chin Ophthal. Res. 13: Florcruz, N.V., and I. Jr.Peczon. 23 Jan Medical interventions for fungal keratitis. Cochrane Database Syst Rev.(1):CD Graf. H., U. Retzke, C. Schilling, and M. Schmidt Reaction of the anterior eye segment to preventive Credé treatment. Zentralbl Gynakol. 116: Klasen, H. J Historical review of the use of silver in the treatment of burns. I. Early uses. Burns 26:

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9 Wang, L. Y., S. T. Sun, L. Zhu, Y. Q. Zhang, Y. Q. Wang, J. C. Li, and J. Xu The pathogenic spectrum investigation of fungal keratitis in 1996~2002 of Henan. Chin J Pract Ophthalmol. 21: Wang, L. Y., Y. Q. Zhang, Y. Q. Wang, G. S. Wang, J. B. Lu, and J. H. Deng Spectrum of mycotic keratitis in China. Chin J Ophthalmol. 36: Wei, J. C Aspergillus Micheli ex Fr. p In J. C. Wei (ed.), Identification Manual of fungi, 1th ed. Scientific & Technologic press, Shanghai. 24. Xie, L. X., X. G. Dong, and W. Y. Shi Treatment of fungal keratitis by penetrating keratoplasty. Br. J. Ophthalmol. 85: Zhang, S. Q Treatment of fungal keratitis with both western medicine and Traditional Chinese medicine. Sichun J Trad Chin Med. 23: Zhong, W. X., S. Y. Sun, J. Zhao, W. Y. Shi, and L. X. Xie Retrospective study of suppurative keratitis in 1054 patients. Chin J Ophthalmol. 43: Table 1. In vitro susceptibilities of ocular Fusarium spp. and Aspergillus spp. isolates to silver nitrate and natamycin Organism(No.) and MICs (µg/ml) antifungal agents Range Mode 50% 90% Members of Fusarium solani species complex (81) silver nitrate natamycin Members of Fusarium moniliforme species complex (19) silver nitrate

10 natamycin Members of Fusarium avenaceum species complex (16) silver nitrate natamycin Members of Fusarium oxysporum species complex (8) silver nitrate natamycin Members of Fusarium poae species complex (4) silver nitrate natamycin Fusarium spp. (128) silver nitrate natamycin Members of Aspergillus flavus species complex (48) silver nitrate natamycin Members of Aspergillus fumigatus species complex (10) silver nitrate natamycin Members of Aspergillus oryzae species complex (12) silver nitrate natamycin Members of Aspergillus versicolor species complex (12) silver nitrate natamycin Members of Aspergillus niger species complex (8) silver nitrate natamycin Aspergillus spp.(90) silver nitrate natamycin