BASIC INVESTIGATION

Comparative Antifungal Susceptibility Analysis of Candida albicans Versus Non-albicans Candida Corneal Isolates Oriel Spierer, MD, Jyoti Dugar, MD, Darlene Miller, DHSc, MPH, CIC, and Terrence P. O’Brien, MD

Purpose: To compare the in vitro activity of topical amphotericin B

(AMB), natamycin, voriconazole, and fluconazole against human corneal isolates of Candida sp. for guidance in the treatment of Candida keratitis.

Methods: Sixty-eight Candida isolates (37 albicans and 31 nonalbicans isolates) recovered from corneal scrapings submitted to rule out microbial keratitis, during the years 2005 to 2011, at the Bascom Palmer Eye Institute, were examined in this study. Corneal isolates were cultured on fungal agars for 48 hours. Each yeast isolate was dispensed into 4 microtiter wells, each containing 100 mL of commercial (natamycin 5%) or compounded (AMB 0.15%, voriconazole 1%, and fluconazole 0.2%) antifungal medications. A comparison of growth patterns was conducted. Results: One hundred percent of the samples showed growth inhibition after treatment exposure with AMB or natamycin. The isolates treated with voriconazole demonstrated an 85% inhibition rate overall, with the Candida albicans samples showing a 77% inhibition rate and the non-albicans sp. a 93% inhibition rate. In the fluconazole group, there was only a 19.6% inhibition rate noted, with a 7.7% inhibition rate observed in the C. albicans group versus a 30% inhibition rate in the non-albicans group. Conclusions: AMB 0.2% and natamycin 5% have equal effectiveness and full inhibition against Candida keratitis isolates. Fluconazole 0.2% is not the drug of choice in both C. albicans and non-albicans keratitis. Voriconazole 1% may need a stronger concentration for higher effectiveness, but potentially may be helpful as a second agent in the treatment of Candida keratitis. Key Words: infectious keratitis, Candida albicans, non-albicans Candida, amphotericin B, natamycin, voriconazole, fluconazole (Cornea 2015;34:576–579)

Received for publication November 19, 2014; revision received December 15, 2014; accepted December 19, 2014. Published online ahead of print March 2, 2015. From the Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, FL. Supported by the Research to Prevent Blindness and by the National Eye Institute core grant P30-EY14801. The authors have no funding or conflicts of interest to disclose. Reprints: Terrence P. O’Brien, MD, Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Palm Beach Gardens, FL 33418 (e-mail: [email protected]). Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

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I

nfectious keratitis is a significant cause of blindness all over the world.1 The incidence of fungal keratitis and the specific pathogens vary greatly by geography. In South Asia, Africa, and other warmer climates, fungal keratitis occurs more frequently than in cooler climates.2 In India, half of corneal ulcers are estimated to be of fungal source.3 In temperate climates, keratitis due to fungi is less common; nevertheless, it can be associated with major morbidity as fungal corneal ulcers typically cause more severe vision impairment than ulcerative keratitis caused by bacterial pathogens.4 Fungal keratitis can result in corneal melting,5 progression to endophthalmitis,6 and phthisis bulbi.5 While in general, filamentous keratitis such as those due to Fusarium and Aspergillus are much more frequent, in temperate climates, such as in the United States, it is believed that yeasts, and especially Candida sp, have a major role in causing fungal keratitis.4,7–10 A survey of over 2 decades in our institute found that Candida sp (most are albicans) accounted for 16.9% of fungal corneal infections.9 Candida is also a pathogen of increasing concern with keratitis in developing countries with poorer outcomes and high rates of need for keratoplasty.5 A recent report from India indicates that Candida is the second major etiologic agent of fungal keratitis, responsible for one fifth of cases of fungal ulcers.5 Known risk factors for fungal keratitis include ocular trauma and contact lens use. Use of topical corticosteroids is another important association.5,11 Corneal graft and chronic ocular surface disease are specific risk factors for development of Candida infections.12 Treatment options for fungal keratitis are limited, and significant variation in management approaches exists between ophthalmologists.8 There is no established gold standard treatment,8 and management of fungal ocular infections remains a challenging and largely empirical endeavor,4 in part because of the expanding list of fungal pathogens and in part because there are few available therapeutic agents. Effective treatment is also compromised because corneal ulcer isolates are not routinely tested by the laboratory for fungi.9 The role of susceptibility testing in guiding treatment decisions is uncertain.4 Current treatments include polyene antifungal agents such as amphotericin B (AMB) and natamycin, or newer azole agents, including fluconazole and voriconazole. There are not enough data in the literature regarding the susceptibility of these antifungal agents against Candida sp causing corneal infections. Given the common failure of these agents in treating these devastating infections and the relatively high associated corneal transplantation ratio,12 it is crucial to gain knowledge Cornea  Volume 34, Number 5, May 2015

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Cornea  Volume 34, Number 5, May 2015

of susceptibility patterns among yeasts causing keratitis. The aim of this study was to compare the in vitro activity of commonly used antifungal treatments, AMB, natamycin, fluconazole, and voriconazole, against corneal isolates of Candida albicans and non-albicans species.

MATERIALS AND METHODS Sixty-eight Candida isolates (37 albicans and 31 nonalbicans isolates) recovered from corneal scrapings submitted to rule out microbial keratitis, during the years 2005 to 2011, at the Bascom Palmer Eye Institute, were examined in this study. The non-albicans isolates included 19 isolates of C. parapsilosis, 7 isolates of C. glabrata, 4 isolates of C. Tropicalis, and 1 isolate of C. lusitaniae. All corneal samples were cultured on Remel Sabouraud dextrose agar (Sab Dex), Remel potato dextrose plate and were also suspended in Remel brain heart infusion broth. Cultures were incubated at a temperature of 30°C for 48 hours. A standard inoculum containing 106 colony forming units per milliliter was prepared by inoculating 2 to 3 colonies from each isolate into 100 mL of yeast nitrogen broth and adjusted using a nephelometer. Ten microliters of each yeast isolate was dispensed into 4-mL wells, each containing 100 mL of commercial (natamycin 5%) or compounded (AMB 0.15%, voriconazole 1%, and fluconazole 0.2%) antifungal medications. Each isolate was also added to a yeast nitrogen broth containing no drug, used as positive controls, whereas yeast nitrogen broth without yeast was used as negative and sterility controls. Microtiter plates were incubated at 30°C and monitored for growth (turbidity) or inhibition (no growth, clear) at 48 hours. Wells with no visible growth were inoculated onto new Sabouraud agar and incubated at 30°C for an additional 48 hours. A comparison of antifungal profiles, which were determined by growth patterns, was performed for the C. albicans isolates and for the nonalbicans isolates.

RESULTS Initial growth was observed for 32 C. albicans corneal isolates and for 30 non-albicans isolates. The other isolates displayed no growth or minimal growth only. After 48 hours of inoculation, 26 C. albicans samples and 30 non-albicans samples showed growth. Six C. albicans isolates either had no growth or only minimal growth. Forty-eight hours after the samples were mixed with antibiotics, they were examined for growth inhibition. Overall, 100% of the samples showed growth inhibition after treatment with AMB 0.15% or with natamycin 5%. The isolates treated with voriconazole 1% demonstrated an 85% inhibition rate overall, with the C. albicans samples showing a 77% inhibition rate (20/26 corneal isolates) and the non-albicans a 93% inhibition rate (28/30 corneal isolates). In the fluconazole 0.2% group, there was only a 19.6% inhibition rate noted, with a 7.7% inhibition rate in the C. albicans group (2/26 corneal isolates) versus a 30% inhibition rate in the non-albicans group (9/30 corneal isolates). In the control group, 100% of samples (56/56 corneal isolates) demonstrated growth. No visible organisms Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.

C. albicans Versus Non-albicans Candida Isolates

were recovered from the wells with no growth after incubation for an additional 48 hours.

DISCUSSION Existing treatment protocols for mycotic infections are far from optimal, because of a combination of delayed diagnosis, growth characteristics of fungi, lack of effective antifungal agents, and limited tissue penetration of the currently available therapeutic agents.2 Fungal keratitis is often refractory to medical treatment, slow to resolve, and frequently requires surgical intervention.13 Current treatments include AMB, natamycin, fluconazole, and voriconazole. In this study, the polyene antifungal agents AMB and natamycin demonstrated equal effectiveness and full inhibition in vitro against C. albicans and non-albicans corneal isolates. The azoles group was observed to be less effective, with the newer azole, voriconazole, being more effective than the older-generation fluconazole against both C. albicans and non-albicans keratitis. Our study is the first to compare the 4 most commonly applied topical medications for treating keratitis caused by Candida sp. Conflicting data regarding the effectiveness of different antifungal drugs exists in the literature. A recent Cochrane Database systematic review of medical interventions for mycotic keratitis analyzed 9 randomized controlled trials involving 568 participants who were randomized to different antifungal drugs. The authors concluded that based on the available studies, there is no evidence that any particular drug, or combination of drugs, is more effective in the management of fungal keratitis.14 AMB, a polyene, is a broad-spectrum antifungal agent.15 It is effective topically for most cases of Candida and other fungi15 and is frequently the treatment of choice for Candidal keratitis.16 AMB has a large molecular weight and is highly insoluble. Solubilizing agents such as desoxycholate (a bile salt) can add to the intrinsic ocular cytotoxicity of this antifungal agent. It does not penetrate well into the corneal stroma,17 and scraping the corneal epithelium is sometimes needed to enhance penetration into the stromal tissues and aqueous humor.18 One study reported 100% sensitivity of AMB for Candida sp.9 However, Sengupta et al5 reported 100% therapeutic failure with AMB, as 16 patients treated with AMB because of C. albicans keratitis required therapeutic keratoplasty, emphasizing the difficulties in extrapolating the in vitro activity to an in vivo effect. Natamycin, another polyene antifungal agent, is the only Food and Drug Administration–approved and commercially available ophthalmic topical antifungal agent.2 It is a standard topical treatment for Candida keratitis7 and is well tolerated.19 However, it has limited solubility and is available only as a 5% suspension. Because of limited corneal penetration, epithelial debridement is sometimes required to achieve sufficient therapeutic tissue concentrations.18 High cost is another limitation.20 The Mycotic Ulcer Topical Treatment Trial (MUTTT) found that natamycin has good clinical and microbiological outcomes.21 However, others reported a primary treatment failure rate of 31% with natamycin.22 www.corneajrnl.com |

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Spierer et al

Cornea  Volume 34, Number 5, May 2015

Fluconazole and its derivate, voriconazole, are both triazoles.23 Topical fluconazole penetrates well into the cornea17 and is reported to be effective in the treatment of corneal ulcers caused by Candida.11 Voriconazole is a relatively new broad-spectrum antifungal agent that was proven to be effective against yeasts and filamentous corneal infections, including Candida sp, Fusarium, and Aspergillus sp.17,24 Corneal penetration of topically administered voriconazole is very good and independent of the state of the epithelial surface, achieving therapeutic aqueous concentrations for Candida sp.13,17,24 Voriconazole eye drops are also well tolerated by patients demonstrating little cytotoxicity.13 Fluconazole resistance among Candida isolates is increasing with some cross-resistance to the new azoles including voriconazole. Resistance is usually dose dependent and occurs more often in the non-albicans.25 Topical AMB is most probably the mainstay for Candida-related keratitis.12 A survey that addressed the actual and preferred treatment of fungal ulcers among 92 cornea specialists found that AMB was the most commonly used treatment for ulcers caused by yeast, followed by natamycin and voriconazole. However, voriconazole was most preferred as the ideal treatment, followed by AMB and natamycin. The main reasons for not using voriconazole in practice, despite it being the preferred choice, were high cost and lack of enough evidence for effectiveness in fungal keratitis. In that survey, approximately half of the cornea specialists listed combination topical therapy as their regimen of choice to treat fungal keratitis, whereas the rest preferred monotherapy.8 According to our results, it seems that the 2 most common treatments, AMB and natamycin, are also the best available treatments, and the presumed superiority of voriconazole is yet to be proven. Furthermore, the MUTT Group21 studied the effect of topical antifungal drops in the treatment of fungal keratitis. The study found that natamycin is superior to voriconazole in terms of visual acuity and likelihood of corneal perforation and need for therapeutic corneal transplantation. The authors concluded that voriconazole should not be used as monotherapy in fungal keratitis.21 Our findings agree with the MUTT findings; however, it should be noted that the MUTT studied filamentous rather than yeast keratitis. Other reports found voriconazole to be more effective than other antifungal medications.9,26 In vitro isolates from Candida keratitis showed that sensitivity was 100% for both AMB and voriconazole.9 Bunya et al26 reported 9 patients with fungal keratitis including C. albicans refractory to AMB, natamycin, and fluconazole, for which their infection was managed with voriconazole. The relatively small sample size is one of the limitations of this study. In addition, the protocol in the study that used a single shot of the antifungal agent is different than real-life treatment that includes much more frequent instillation of antifungal drops. Also, we did not determine susceptibility as minimal inhibitory concentration or minimal cidal concentration, which may be useful as they can be compared to likely obtained cornea levels. Finally, the nature of the study, which is an in vitro comparison, may not fully reflect the in vivo activity. Nevertheless, in vitro growth patterns and susceptibility to antimicrobial drugs are a primary guide (with

1. Whitcher JP, Srinivasan M, Upadhyay MP. Corneal blindness: a global perspective. Bull World Health Organ. 2001;79:214–221. 2. Hariprasad SM, Mieler WF, Lin TK, et al. Voriconazole in the treatment of fungal eye infections: a review of current literature. Br J Ophthalmol. 2008;92:871–878. 3. Srinivasan M, Gonzales CA, George C, et al. Epidemiology and aetiological diagnosis of corneal ulceration in Madurai, south India. Br J Ophthalmol. 1997;81:965–971. 4. Lalitha P, Prajna NV, Oldenburg CE, et al. Organism, minimum inhibitory concentration, and outcome in a fungal corneal ulcer clinical trial. Cornea. 2012;31:662–667. 5. Sengupta J, Khetan A, Saha S, et al. Candida keratitis: emerging problem in India. Cornea. 2012;31:371–375. 6. Henry CR, Flynn HW Jr, Miller D, et al. Infectious keratitis progressing to endophthalmitis: a 15-year study of microbiology, associated factors, and clinical outcomes. Ophthalmology. 2012;119:2443–2449. 7. Tanure MA, Cohen EJ, Sudesh S, et al. Spectrum of fungal keratitis at Wills Eye Hospital, Philadelphia, Pennsylvania. Cornea. 2000;19: 307–312. 8. Loh AR, Hong K, Lee S, et al. Practice patterns in the management of fungal corneal ulcers. Cornea. 2009;28:856–859. 9. Marangon FB, Miller D, Giaconi JA, et al. In vitro investigation of voriconazole susceptibility for keratitis and endophthalmitis fungal pathogens. Am J Ophthalmol. 2004;137:820–825. 10. Pouyeh B, Galor A, Miller D, et al. New horizons in one of ophthalmology’s challenges: fungal keratitis. Expert Rev Ophthalmol. 2011;6:529–540. 11. Matsumoto Y, Murat D, Kojima T, et al. The comparison of solitary topical micafungin or fluconazole application in the treatment of Candida fungal keratitis. Br J Ophthalmol. 2011;95:1406–1409. 12. Sun RL, Jones DB, Wilhelmus KR. Clinical characteristics and outcome of Candida keratitis. Am J Ophthalmol. 2007;143:1043–1045. 13. Lau D, Fedinands M, Leung L, et al. Penetration of voriconazole, 1%, eyedrops into human aqueous humor: a prospective open-label study. Arch Ophthalmol. 2008;126:343–346. 14. FlorCruz NV, Peczon IV, Evans JR. Medical interventions for fungal keratitis. Cochrane Database Syst Rev. 2012;15:CD004241. 15. Mahdy RA, Nada WM, Wageh MM. Topical amphotericin B and subconjunctival injection of fluconazole (combination therapy) versus topical amphotericin B (monotherapy) in treatment of keratomycosis. J Ocul Pharmacol Ther. 2010;26:281–285. 16. Thomas PA. Fungal infections of the cornea. Eye (Lond). 2003;17: 852–862. 17. Al-Badriyeh D, Leung L, Davies GE, et al. Successful use of topical voriconazole 1% alone as first-line antifungal therapy against Candida albicans keratitis. Ann Pharmacother. 2009;43:2103–2107.

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clinical judgment) in making clinical treatment decisions for infective keratitis. In conclusion, this comparative in vitro study found that AMB 0.15% and natamycin 5% have equal effectiveness and full inhibition against C. albicans and non-albicans keratitis isolates. The newer azole, voriconazole 1%, proves to be more effective than the older-generation fluconazole 0.2% against both C. albicans and non-albicans keratitis. This indicates some emerging resistance to fluconazole and suggests that fluconazole is not the drug of choice in both C. albicans and non-albicans keratitis. According to our results, it seems that voriconazole 1% should not be used as monotherapy; nevertheless, it may be helpful as a second agent in the treatment of keratitis caused by Candida. A statement regarding the in vivo superiority of one drug or the other in the treatment of yeast keratitis can only be propagated when a prospective comparative randomized therapy trial is conducted for yeast keratitis. REFERENCES

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18. O’Day DM, Head WS, Robinson RD, et al. Corneal penetration of topical amphotericin B and natamycin. Curr Eye Res. 1986;5: 877–882. 19. Thomas PA, Kaliamurthy J. Mycotic keratitis: epidemiology, diagnosis and management. Clin Microbiol Infect. 2013;19:210–220. 20. Carrasco MA, Genesoni G. Treatment of severe fungal keratitis with subconjunctival amphotericin B. Cornea. 2011;30:608–611. 21. Prajna NV, Krishnan T, Mascarenhas J, et al; Mycotic Ulcer Treatment Trial Group. The mycotic ulcer treatment trial: a randomized trial comparing natamycin vs voriconazole. JAMA Ophthalmol. 2013;131: 422–429. 22. Lalitha P, Prajna NV, Kabra A, et al. Risk factors for treatment outcome in fungal keratitis. Ophthalmology. 2006;113:526–530.

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C. albicans Versus Non-albicans Candida Isolates

23. Sabo JA, Abdel-Rahman SM. Voriconazole: a new triazole antifungal. Ann Pharmacother. 2000;34:1032–1043. 24. Thiel MA, Zinkernagel AS, Burhenne J, et al. Voriconazole concentration in human aqueous humor and plasma during topical or combined topical and systemic administration for fungal keratitis. Antimicrob Agents Chemother. 2007;51:239–244. 25. Pappas PG, Kauffman CA, Andes D, et al; Infectious Diseases Society of America. Clinical practice guidelines for the management of candidiasis: 2009 update by the Infectious Diseases Society of America. Clin Infect Dis. 2009;48:503–535. 26. Bunya VY, Hammersmith KM, Rapuano CJ, et al. Topical and oral voriconazole in the treatment of fungal keratitis. Am J Ophthalmol. 2007; 143:151–153.

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Comparative antifungal susceptibility analysis of Candida albicans versus non-albicans Candida corneal isolates.

To compare the in vitro activity of topical amphotericin B (AMB), natamycin, voriconazole, and fluconazole against human corneal isolates of Candida s...
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