CLINICAL SCIENCE

Factors Affecting Treatment Outcomes With Voriconazole in Cases With Fungal Keratitis
Thushanthi Ramakrishnan, MBBS,* Marios Constantinou, BSc (Hons), BOrth, Vishal Jhanji, MD, and Rasik B. Vajpayee, MBBS, MS, FRCSEd, FRANZCO*

Purpose: To report clinical experience with the use of voriconazole

for management of cases with fungal keratitis and to evaluate the factors affecting treatment outcomes.

Methods: Retrospective database review of all cases with fungal

keratitis that were treated with topical voriconazole at the Royal Victorian Eye and Ear Hospital, Melbourne, Australia, between January 2003 and July 2010, was undertaken. Main parameters evaluated were clinical and microbiological prole of the cases, treatment, and nal outcomes.

Results: A total of 26 cases were treated with voriconazole eye drops during the study period. In addition, voriconazole was used in the form of oral tablets (n = 16, 61.5%), intracorneal injection (n = 7, 26.9%), and intracameral injection (n = 2, 7.7%). Overall, 50% (n = 13) of cases responded to medical treatment. Further denitive surgical intervention in the form of penetrating keratoplasty was required in 11 cases (42.3%). Two cases (7.7%) underwent enucleation for severe nonresolving keratitis. Nonresponders were more likely to have peripheral inltrates (38.5% vs. 7.7%, P = 0.16) and hypopyon (61.5% vs. 23%, P = 0.11) as compared with responders. There was no signicant variation in the microbiological results between responders and nonresponders. Conclusions: Voriconazole was successful in the management of 50% cases of fungal keratitis in our study. Cases with peripheral inltrates and hypopyon are less likely to respond to medical treatment.
Key Words: fungal keratitis, voriconazole, management, outcomes (Cornea 2013;32:445449)

ungal keratitis is recognized as a signicant cause of ocular morbidity and blindness. Although the incidence and etiology of fungal keratitis vary considerably with geographical location, the overall prevalence has demonstrated an upward trend in recent years.15 This may be attributed to several factors including frequent and chronic use of topical corticosteroids and contact lenses, and sensitivity of diagnostic techniques.3,6 Fungal keratitis remains a therapeutic challenge to ophthalmologists because of a lack of an established gold standard treatment. Most of the challenges in managing these patients are as a result of delay in making the correct diagnosis and the limited efcacy of existing antifungal therapies. Voriconazole is a new-generation triazole and its successful use, both systemically and topically in the management of fungal keratitis, has renewed interest with promising reports in medical literature.7 Clinical and in vitro studies of voriconazole have demonstrated high potency with lower minimum inhibitory concentrations, high tissue penetration, and a broad spectrum of activity when compared with other antifungal agents.79 These properties suggest that voriconazole may emerge as a potential rst-line agent for the management of fungal keratitis. In the present study, we report the outcomes of cases with fungal keratitis that were treated with voriconazole at our hospital. We also endeavored to analyze the factors associated with failure of treatment response in these cases.

METHODS
A retrospective database review was undertaken for all patients who were treated with topical voriconazole at the Royal Victorian Eye and Ear Hospital, Melbourne, Australia, between January 2003 and July 2010. Cases were identied from the database of pharmacological codes, eliciting all patients with fungal keratitis who had been treated with topical voriconazole. Charts were reviewed to determine whether fungal keratitis was culture proven, and only those that were culture proven were included in the analyses presented here. The study followed the tenets of Declaration of Helsinki and was approved by the Human Research Ethics Committee of the hospital. At initial presentation, all patients underwent a detailed ophthalmic assessment, including clinical history, best-corrected visual acuity, and slit-lamp biomicroscopy. Corneal scrapings were obtained for microbiological evaluation before initiation of any treatment. The corneal specimens were inoculated onto
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Received for publication October 27, 2011; revision received January 20, 2012; accepted March 2, 2012. From the *Royal Victorian Eye and Ear Hospital, Melbourne, Victoria, Australia; Centre for Eye Research Australia, Melbourne University Department of Ophthalmology, Australia; and Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China. Supported by Victorian Governments Operational Infrastructure Support to the Centre for Eye Research Australia. Presented at the annual meeting of the Association for Research in Vision and Ophthalmology 2011. The authors state that they have no proprietary interest in the products named in this article. Reprints: Rasik B. Vajpayee, Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, 32 Gisborne St, Melbourne, Victoria 3002, Australia (e-mail: rasikv@unimelb.edu.au). Copyright 2012 by Lippincott Williams & Wilkins

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blood agar, chocolate agar, Sabouraud dextrose agar, and thioglycollate broth. Two glass slides were prepared for Gram stain and for Blankophor stain, and a swab was taken for detection of herpes virus by polymerase chain reaction. Subsequently, intensive antimicrobial therapy was started in the form of hourly ooxacin 0.3% eye drops. All patients with culture-proven fungal keratitis were commenced on hourly topical antifungal therapy (natamycin 5%, amphotericin 5%, or voriconazole 1%). Oral voriconazole (2 loading doses of 400 mg, followed by 200 mg twice daily) was commenced at the discretion of the treating ophthalmologist when deemed necessary. The treatment was modied as indicated by culture results, susceptibility pattern, and clinical response. Successful treatment was dened by complete resolution of corneal infection. Patients who did not respond to topical natamycin or amphotericin were commenced on topical voriconazole. Ooxacin eye drops were continued in cases with mixed infections. Progression of the disease despite intensive medical management, corneal perforation or impending perforation, was an indication for surgical intervention. Data reviewed included age, sex, predisposing factors (ocular and systemic), clinical presentation and treatment, visual outcomes, microbiological prole, and surgical interventions. We divided the cases into responders and nonresponders based on the response to voriconazole therapy. Further analysis was performed to report any signicant difference between these 2 groups. All statistical analyses were performed using the Statistical Package for the Social Sciences for Windows (version 12.0; SPSS Inc, Chicago, IL). Results were expressed as mean SD. Fisher exact test was used for categorical outcomes and t tests were used for comparing continuous variables, with a P value ,0.05 considered signicant.

TABLE 1. Microbiological Culture Results in Responder and Nonresponder Groups in Cases With Fungal Keratitis
Total Bacterial isolates Staphylococcus aureus Staphylococcus epidermidis Coagulase-negative Staphylococcus Pseudomonas aeruginosa Micrococcus species Mycobacterium chelonae Corynebacterium species Streptococcus viridans Streptococcus oralis Bacillus species Propionibacterium species Enterococcus faecalis Fungal isolates Fusarium species Candida albicans Candida parapsilosis Scedosporium apiospermum Aspergillus fumigatus Paecilomyces species Bipolaris species Penicillium species Alternaria species Phaeoacremonium species Others Acanthamoeba Herpes simplex virus 3 3 2 2 2 2 1 1 1 1 1 1 7 2 2 3 3 2 2 1 1 1 1 1 Responders 2 2 2 0 2 0 0 0 0 1 0 1 3 1 1 1 2 0 2 0 0 1 1 0 Nonresponders 1 1 0 2 0 2 1 1 1 0 1 0 4 1 1 2 1 2 0 1 1 0 0 1

RESULTS
A total of 26 patients (15 men, 11 women) were treated with voriconazole eye drops for fungal keratitis between January 2003 and July 2010. Mean age was 58.1 15.9 years (range: 20.585.4 years), and average duration between onset of symptoms and initiation of antifungal therapy was 11.8 12.6 days (range: 155 days). Mean visual acuity (decimal) at the time of presentation was 0.18 0.27 (range: 0.50.001). Predisposing risk factors identied included a history of ocular surgery (n = 7, 27%), trauma (n = 6, 23%), ocular surface disease (n = 6, 23%), and contact lens use (n = 3, 12%). Sixteen patients (61.5%) were using long-term topical (n = 15) or systemic (n = 1) corticosteroids at the time of presentation. Before presentation to our hospital, 7 cases (27%) had received treatment for bacterial keratitis and 7 (27%) were being treated for herpes simplex virus (HSV) keratitis. On slit-lamp examination, all cases had central (n = 16, 61.5%), midperipheral (n = 4, 15.4%), or peripheral (n = 6, 23%) corneal inltrates. Eleven cases (42.3%) presented with a hypopyon. Fusarium species was the most commonly isolated fungus (n = 7, 27%), followed by Candida species (n = 4, 15.4%; Table 1). Sixteen cases (61.5%) had a microbial coinfection most commonly with Staphylococcus (n = 8, 30.8%).

Coinfections with HSV (n = 1, 3.8%) and Acanthamoeba (n = 1, 3.8%) were observed in 1 case each. Antifungal eye drops were added after the microbiological results of corneal scrapings were received. Initial antifungal treatment consisted of topical natamycin (n = 6, 23%), amphotericin B (n = 3, 11.5%), or voriconazole (n = 17, 65.3%). Although topical voriconazole was used initially in 17 cases, voriconazole was eventually added in the other cases. Therefore, topical voriconazole was used in all cases included in this study (Table 2). Voriconazole was used in the form of oral tablets (n = 16, 61.5%), intracorneal injection (n = 7, 26.9%), and intracameral injection (n = 2, 7.7%). Oral voriconazole was used for a mean duration of 50.4 62.7 days (range: 10163 days). Topical antifungal drops were administered for a mean duration of 57.2 78.0 days (range: 15298 days). Complete resolution of infection was achieved in 13 cases (50%). Mean visual acuity of 0.63 0.38 (decimal) was recorded in these cases, with variable corneal scarring on nal review. Further surgical intervention in the form of penetrating keratoplasty was performed in 11 cases (42.3%), and 2 patients (7.7%) subsequently required an enucleation for severe nonresolving fungal keratitis. One of these patients was a 63-year-old woman with metastatic breast cancer who was undergoing chemotherapy at the time of presentation.
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TABLE 2. Summary of Cases Based on Response to Voriconazole Treatment for Fungal Keratitis
Case 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Initial Topical Treatment Voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Natamycin Natamycin Natamycin Natamycin Natamycin Natamycin Amphotericin B Amphotericin B Amphotericin B Voriconazole Voriconazole Added Topical Treatment Natamycin Adjunct Treatment Intrastromal voriconazole Final Outcome Enucleation Healed Keratoplasty Keratoplasty Keratoplasty Healed Healed Healed Enucleation Keratoplasty Healed Keratoplasty Healed Healed Keratoplasty Healed Healed Keratoplasty Keratoplasty Keratoplasty Healed Keratoplasty Healed Healed Healed Keratoplasty

Intrastromal voriconazole

Natamycin, amphotericin B Natamycin Natamycin Natamycin Natamycin, amphotericin B Voriconazole Voriconazole Voriconazole Amphotericin B, voriconazole Voriconazole Voriconazole Voriconazole Voriconazole Natamycin, voriconazole

Intrastromal voriconazole Intrastromal voriconazole

Intracameral voriconazole Intrastromal voriconazole Intrastromal voriconazole Intracameral voriconazole

Initial microbiological evaluation identied Pseudomonas aeruginosa and HSV from the corneal scrapings. Despite intensive treatment with topical ooxacin 0.3% drops and oral acyclovir, progression of the corneal ulcer resulted in a corneal perforation. A penetrating keratoplasty was undertaken and evaluation of the corneal button identied Aspergillus fumigatus. Topical natamycin, topical voriconazole, and intrastromal voriconazole were added. However, the infection spread to the sclera and the patient proceeded to have an enucleation. The second patient was an 83-year-old man who presented with keratitis and melting scleral graft that was undertaken for postbeta irradiation scleromalacia 10 years ago. Scedosporium apiospermum was isolated from the corneal scrapings. Management included topical, oral, and intrastromal voriconazole. The disease however progressed to sclerokeratitis, and subsequently, the patient required an enucleation. We performed further analysis based on the success or failure of voriconazole treatment in our patients. Overall, there were no statistically signicant differences in the clinical and microbiological characteristics in both groups. Patients who demonstrated complete resolution of fungal infection were younger (50.0 15.8 years vs. 67.5 12.2 years, P = 0.22), had better visual acuity at the time of presentation (0.31 0.31 vs. 0.07 0.15, P = 0.32), and were diagnosed earlier (10.6 9.8 days vs. 12.9 15.1 days, P = 0.86), as compared with the nonresponders. None of the patients with a history of prior ocular surgery responded (n = 7, 26.9%) to the treatment,
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whereas all patients with prior contact lens use (n = 3) were responders. Ten of 16 patients (62.5%) who were using topical or systemic corticosteroids at the time of presentation did not respond to treatment (P = 0.23; Table 3). Furthermore, mean duration of use of corticosteroid eye drops was longer in nonresponders as compared with responders (39.4 42.3 days vs. 17.8 20.9 days, P = 0.52). A previous diagnosis of bacterial keratitis was more common in the responders (n = 5/13, 38.5%) compared with the nonresponders (n = 2/13, 15.4%, P = 0.38). On further analysis, we found that cases with coexistent bacterial keratitis (n = 7) that responded to treatment (n = 5) presented earlier (6 3.3 days) as compared with those with (n = 2,
TABLE 3. Predisposing Risk Factors in Responders and Nonresponders in Cases With Fungal Keratitis Treated With Voriconazole
Responders Prior ocular surgery Ocular trauma Ocular surface disease Contact lens use Corticosteroid use (topical or systemic) 0 2 2 3 6 Nonresponders 7 4 4 0 10 Total 7 6 6 3 16

No statistically signicant differences were found for any of the risk factors.

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15.3 7.7 days) or without (n = 11, 13.5 16.6 days) bacterial keratitis that did not respond to treatment. Nonresponders were more likely to present with a peripheral inltrate (P = 0.16) and a hypopyon (P = 0.11) as compared with the responders. However, this difference was statistically nonsignicant (Table 4). All cases in which intrastromal or intracameral voriconazole was used ultimately required another surgical intervention in the form of keratoplasty or enucleation. There was no signicant variation in the microbiological results in the 2 groups (Table 1).

DISCUSSION
In the present study, 50% of cases with fungal keratitis responded to treatment with voriconazole eye drops. Further denitive surgical intervention in the form of penetrating keratoplasty was required in 11 cases (42.3%), and 2 cases (7.7%) underwent enucleation for severe nonresolving keratitis. There were no signicant differences in the clinical and microbiological proles of responders and nonresponders. Voriconazole has a broad spectrum of activity against yeast, and dematiaceous and lamentous fungi.10 It is available commercially for systemic administration in the form of oral and intravenous formulations. Oral voriconazole has high bioavailability and demonstrates good penetration into the different parts of the eye,11 with sufcient concentrations achieved to cover a wide range of keratitis-causative fungi. Topical voriconazole eye drops, manufactured extemporaneously and used in an off-label manner, have been prescribed for the treatment of keratitis. A previous study has shown that with topical administration, voriconazole demonstrates good penetration through the cornea into the aqueous humor.12 In the present study, an overall success was achieved in 50% cases treated with voriconazole. The remaining 50% required further surgical intervention mostly in the form of keratoplasty. Therapeutic keratoplasty has a denitive role in the management of fungal corneal ulcers. The major causative agents isolated are Fusarium solani, Aspergillus fumigatus, and Candida albicans.13 The anatomical and functional success rates are inferior in cases with perforated fungal corneal ulcers compared with those with impending perforation.13 Comparative data report the incidence of penetrating keratoplasty for the denitive management of fungal keratitis to be 18% to 29%.13,14 The higher rate for surgical intervention demonstrated

TABLE 4. Clinical Features of Corneal Inltrates at the Time of Presentation in Responders and Nonresponders in Cases With Fungal Keratitis
Clinical Features Location of inltrate Central Midperiphery Periphery Hypopyon present Responders (n = 13) Nonresponders (n = 13) P Total

9 3 1 3

7 1 5 8

0.16 0.59 0.16 0.11

16 4 6 11

in our study may indicate delayed presentation or referral of more severe cases to a tertiary care eye hospital. More than half of the patients (61.5%) in our study were using corticosteroids at the time of presentation. Furthermore, the proportion of these patients was higher in the nonresponders as compared with the responders. Topical steroids have been shown to increase the rate of conjunctival colonization and indirectly promote fungal replication and corneal invasion by interfering with the hosts inammatory response.15 Similarly, systemic steroids promote fungal keratitis by rendering the host immunocompromised.16 History of ocular surgery was present in 26.9% of cases in our study. It is noteworthy that none of these cases responded to medical antifungal treatment. The pathophysiology of fungal keratitis after ocular surgery may be attributed to introduction of the organisms directly via surgery, or a delayed breach of the ocular surface via sutures or abnormal epithelium that overlies incisions.17 Miedziak et al18 also identied a history of ocular surgery and long-term topical steroid use as pertinent risk factors correlating with a need for penetrating keratoplasty in the management of microbial keratitis. We did not nd any statistically signicant differences in the clinical and microbiological features of responders and nonresponders. Cases with prior diagnosis of bacterial keratitis showed a trend toward better response to voriconazole treatment possibly as a result of early presentation. Cases with peripheral corneal inltrates and hypopyon at the time of presentation required surgical intervention more frequently than those without these features. Previous studies have shown that ulcers with these characteristics have poorer outcomes and are more likely to require surgical intervention.19,20 Failure to respond to voriconazole therapy in peripherally located inltrates can be attributed to early scleral involvement in such cases. Extension of the disease into the sclera often translates to an unfavorable prognosis because of poor antimicrobial penetration in the avascular sclera and the ability of the microorganisms to multiply before instigating an inammatory response.19 The presence of a hypopyon in fungal keratitis also indicates invasive and severe disease. Although the presence of a hypopyon in a bacterial keratitis is often sterile, the presence of a hypopyon in fungal keratitis may be because of direct invasion into the anterior chamber of fungal hyphae enmeshed in thick exudates.21 A previous study has reported the presence of large ulcers and hypopyon at the time of presentation as predictors of poor outcomes in cases with fungal keratitis.20 Prajna et al22 have compared the clinical outcomes of treatment with topical natamycin versus topical voriconazole for fungal keratitis in a multicenter, double-masked clinical trial. Rates of corneal perforations were 16.6% and 15% in the voriconazole and natamycin groups, respectively. The authors did not nd any signicant differences in visual acuity and scar size between voriconazoletreated and natamycin-treated patients. In our study, treatment with voriconazole was successful in 50% of cases. Voriconazole was not used as a rst line of treatment in some cases, and it is possible that infection may have already progressed in these cases. Also, oral voriconazole was not used in all cases. Surgical intervention was required in the remaining 50% of cases mainly in the
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7. 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;92:871878. 8. Prakash G, Sharma N, Goel M, et al. Evaluation of intrastromal injection of voriconazole as a therapeutic adjunctive for the management of deep recalcitrant fungal keratitis. Am J Ophthalmol. 2008;146:5659. 9. Al-Badriyeh D, Neoh CF, Stewart K, et al. Clinical utility of voriconazole eye drops in ophthalmic fungal keratitis. Clin Ophthalmol. 2010;4:391405. 10. Marangon FB, Miller D, Giaconi JA, et al. In vitro investigation of voriconazole susceptibility for keratitis and endophthalmitis fungal pathogens. Am J Ophthalmol. 2004;137:820825. 11. Hariprasad SM, Mieler WF, Holz ER, et al. Determination of vitreous, aqueous, and plasma concentration of orally administered voriconazole in humans. Arch Ophthalmol. 2004;122:4247. 12. Klont R, Eggink C, Rijs A, et al. Successful treatment of Fusarium keratitis with cornea transplantation and topical and systemic voriconazole. Clin Infect Dis. 2005;40:e110e112. 13. Sony P, Sharma N, Vajpayee RB, et al. Therapeutic keratoplasty for infectious keratitis: a review of the literature. CLAO J. 2002;28:111118. 14. Foster RK. The role of excisional keratoplasty in microbial keratitis. In: Cavanagh HD, ed. The Cornea Transactions of World Congress on the Cornea. 3rd ed. New York, NY: Raven; 1988:198. 15. Bharathi MJ, Ramakrishnan R, Vasu S, et al. Epidemiological characteristics and laboratory diagnosis of fungal keratitis. A three-year study. Indian J Ophthalmol. 2003;51:315321. 16. Mitsui Y, Hanabusa J. Corneal infections after cortisone therapy. Br J Ophthalmol. 1955;39:244250. 17. Jhanji V, Sharma N, Mannan R, et al. Management of tunnel fungal infection with voriconazole. J Cataract Refract Surg. 2007;33:915917. 18. Miedziak A, Miller MR, Rapuano C, et al. Risk factors in microbial keratitis leading to penetrating keratoplasty. Ophthalmology. 1999;106:11661170. 19. Jhanji V, Yohendran J, Constantinou M, et al. Scedosporium scleritis or keratitis or both: case series. Eye Contact Lens. 2009;35:312315. 20. Lalitha P, Prajna NV, Kabra A, et al. Risk factors for treatment outcome in fungal keratitis. Ophthalmology. 2006;113:526530. 21. Ramsay A, Lightman S. Hypopyon uveitis [Review]. Surv Ophthalmol. 2001;46:118. 22. Prajna NV, Mascarenhas J, Krishnan T, et al. Comparison of natamycin and voriconazole for the treatment of fungal keratitis. Arch Ophthalmol. 2010;128:672678.

form of corneal transplantation. Two patients underwent enucleation for extensive nonresolving fungal infection. The refractory keratitis in both cases may be attributed to extensive disease with scleral involvement. Furthermore, one of these patients was also severely immunocompromised and was receiving concomitant chemotherapy. Management of fungal keratitis remains a therapeutic challenge. In the present study, although we did not nd any statistically signicant differences between responders and nonresponders, this may be attributed to the small sample size and the retrospective nature of the study. Voriconazole is increasingly used in clinical practice to treat fungal keratitis mostly on the basis of in vitro and anecdotal results. Similar studies with a prospective design and a larger sample size would further encourage the use of voriconazole in suitable cases of fungal keratitis. Also, future clinical trials would be necessary to characterize factors determining its success in the management of cases with fungal keratitis. REFERENCES
1. Gower EW, Keay LJ, Oechsler RA, et al. Trends in fungal keratitis in the United States, 2001 to 2007. Ophthalmology. 2010;117:22632267. 2. Saha R, Das S. Mycological prole of infectious keratitis from Delhi. Indian J Med Res. 2006;123:159164. 3. Gopinathan U, Garg P, Fernandes M, et al. The epidemiological features and laboratory results of fungal keratitis: a 10-year review at a referral eye care center in South India. Cornea. 2002;21:555559. 4. Shokohi T, Nowroozpoor-Dailami K, Moaddel-Haghighi T. Fungal keratitis in patients with corneal ulcer in Sari, Northern Iran. Arch Iran Med. 2006;9:222227. 5. Leck AK, Thomas PA, Hagan M, et al. Aetiology of suppurative corneal ulcers in Ghana and south India, and epidemiology of fungal keratitis. Br J Ophthalmol. 2002;86:12111215. 6. Shukla PK, Kumar M, Keshava GB. Mycotic keratitis: an overview of diagnosis and therapy. Mycoses. 2008;51:183199.

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