Abstract
Objective
To determine the efficacy of adjunctive topical tacrolimus in improving graft survival and reducing post operative complications in patients with Therapeutic Penetrating Keratoplasty (TPK) for the treatment of fungal keratitis.
Study design
Prospective consecutive series of 13 patients at Aravind Eye Hospital in Pondicherry, India with smear/culture positive fungal keratitis who require TPK
Main outcome measures
The primary outcome was graft clarity at 6 months. The secondary outcomes included BSCVA at 3 months and 6 months as well as intra-operative and post-operative complications.
Results
A total of 13 patients with smear/culture positive fungal keratitis who underwent TPK were enrolled. The mean age of the patients was 62 years, with 77 % being male. Microbiological evidence of fungal infection was present in all patients. 9 out of 13 patients (69 %) were determined to either have a good outcome or be a good candidate for subsequent optical penetrating keratoplasty (PKP). BSCVA improved by 3.5 lines. Three patients had reinfection and required repeat TPK. There were no cases of endophthalmitis, and no patients required urgent glaucoma surgery.
Conclusions
Adjunctive topical tacrolimus after therapeutic penetrating keratoplasty may reduce post operative complications and improve the chances of subsequent successful optical penetrating keratoplasty.
Clinicaltrials gov identifier
1. Introduction
Tacrolimus (FK506) has both anti-inflammatory and anti-fungal properties making it a compelling potential therapy in the treatment of therapeutic penetrating keratoplasty for fungal keratitis.1,2 It is a potent immunosuppressant of T-cell proliferation through inhibition of Calcineurin signaling pathways and has been used systemically to prevent solid organ transplant rejection and topically to treat allergic dermatitis and keratoconjunctivitis. FK506 has also been shown to have broad antifungal activity in vitro, which may be related to the fact that fungi rely on the calcineurin pathway for growth and virulence.3 Of particular interest is the fact that FK506 may be able to increase efficacy of topical antifungals or even reverse resistance.
There is an urgent need for novel therapeutics to improve outcomes of fungal keratitis, a major cause of blindness worldwide. Topical natamycin, a polyene, is the only antifungal agent approved in the United States by the Food and Drug Administration (FDA) for treatment of fungal keratitis. The Mycotic Ulcer Treatment Trials (MUTT) I and II, were NEI-funded randomized double-masked clinical trials that found topical natamycin to be superior to topical voriconazole and no additional benefit of adjuvant oral voriconazole. Two recent randomized clinical trials also failed to demonstrate a benefit of adjuvant intrastromal voriconazole or adjuvant corneal crosslinking in the treatment of fungal keratitis.4,5 However, natamycin is fungistatic and has limited penetration into the corneal layers.6 Furthermore, outcomes of fungal keratitis with topical natamycin are extremely poor as demonstrated in MUTT II where approximately 50 % of patients had full thickness corneal perforation or required therapeutic penetrating keratoplasty (TPK) despite topical natamycin, topical voriconazole, and adjunctive oral voriconazole.
The host response to fungal infection is a complex process that involves the release of cytokines like interleukins, Tumour Necrosis Factor (TNF), and chemokines, which recruit macrophages and neutrophils to the infection site, initiating subsequent inflammatory reactions. While the release of inflammatory mediators is crucial for eliminating fungal pathogens, an excessive inflammatory response can also damage healthy corneal tissue. Hence, achieving a balance between effective fungal clearance and minimizing tissue damage is essential.7,8
Topical tacrolimus 0.1 % may be able to strike the delicate balance between reducing host inflammatory responses without increasing the recurrence of infection. Here we describe a small series of patients treated with adjunctive topical tacrolimus therapy after TPK for filamentous fungal ulcers.
2. Methods
2.1. Study population
In this prospective consecutive series, patients presenting to the Aravind Eye Hospital at Pondicherry with either smear or culture positive fungal corneal ulcers requiring therapeutic penetrating keratoplasty, were screened for inclusion. Baseline demographic data were prospectively collected and ulcer characteristics including onset of symptoms, prior antimicrobial therapy, duration of treatment, organism, ulcer size were also recorded.
2.2. Surgery
Therapeutic corneal transplantation (TPK) with optical corneal graft tissue was performed at a single site by three experienced corneal surgeons (JC, KM & RB) following standard institutional protocols for TPK. The corneal tissue used was of optical quality with a minimum endothelial count of 2000 cells/mm4 and minimum death to surgery time of 11 days. Concurrent lens removal was performed as needed. Intracameral voriconazole was administered at the end of the case.
2.3. Intervention
On the first post-operative day, study participants began topical natamycin 5 % (Natamet, Sunpharma, Mumbai, India) every hour for 2 days followed by 4 times daily for 1 month. They were also treated with topical Moxifloxacin 0.05 % (Milflox, Sunpharma, Mumbai, India) 4 times daily for 2 weeks or until epithelial healing, and homide 2 % (Indoco, Mumbai, India) drops twice daily. Tacrolimus 0.1 % ointment (Ajanta, Mumbai, India) was started 3 times daily for 2 months and then tapered to 2 times daily for 2–3 months. Thereafter the patient was instructed to use the tacrolimus daily at bedtime until 6 months post-op. Prednisolone acetate 1 % (Predmet Sunpharma, Mumbai, India) was started after 1 month if there was no evidence of re-infection. Prednisolone was continued QID for 3 months, TID for 3 months, BID until 6 months and daily until 1 year post-op.
2.4. Outcomes
The primary study outcome was graft clarity at 6 months. Graft failure defined as irreversible moderate corneal edema or opacity. Secondary outcomes included BSCVA at 3 and 6 months, preservation of the anterior chamber anatomy, and complications including recurrence of infection, development of glaucoma or need for further surgery.
2.5. Statistical analysis
Baseline demographic and ulcer characteristics will be outlined using descriptive statistics. A paired two-sided t-test was done to estimate the mean difference in BSCVA between baseline and 6-month.
3. Results
A total of 13 patients with smear and/or culture positive fungal keratitis requiring TPK were enrolled. Table 1 outlines the baseline characteristics. Seventy-seven percent (N = 10) were male, with a mean age of 62 years (SD 10). The reason for TPK was corneal perforation in 4/13 (30 %), impending perforation in 2/13 (15 %), progression to the limbus and/or worsening infection despite maximal medical therapy in 7/13 (54 %). All patients had poor visual acuity at baseline with a mean logMAR visual acuity of 2.2 (SD 0.05) and mean corneal ulcer size was 6.3 mm (SD 1.3). All patients had microbiological evidence of filamentous fungus either on corneal smear (N = 11, 85 %), corneal culture (N = 12, 92 %) and/or culture of the corneal button (N = 7, 54 %). Aspergillus spp (N = 5, 38 %) and Fusarium spp (N = 5, 38 %) were the most commonly isolated fungi (Table 2).
Table 1.
Baseline demographic features and medications.
| Overall (N = 13) | ||
|---|---|---|
| Sex | Male | 10 (77 %) |
| Age (years) | Mean | 62 (10) |
| Symptom Duration (days) | Mean | 7.6 (7.6) |
| BCVA (log MAR) | HM | 10 (77 %) |
| Epithelial Defect Size (mm) | Mean (SD) | 3.4 (3.5) |
| Corneal Ulcer Size (mm) | Mean (SD) | 6.3 (1.3) |
| Natamycin | Yes | 7 (54 %) |
| Moxifloxacin | Yes | 4 (31 %) |
| Voriconazole | Yes | 2 (15 %) |
| Atropine | Yes | 2 (15 %) |
| Gemifloxacin | Yes | 2 (15 %) |
| Duration of medical treatment (days) | Mean (SD) | 23 (23) |
Table 2.
Baseline corneal and corneal button smear and culture results.
| Overall (N = 13) | ||
|---|---|---|
| Fungal Culture/ Smear/Corneal Button | Positive culture or smear or corneal button | 12 (92 %) |
| Fungal Smear | Positive | 11 (85 %) |
| Fungal Culture | Positive | 11 (85 %) |
| Corneal button growth | Yes | 7 (54 %) |
| Fungal Organism | Aspergillus flavus | 4 (31 %) |
| Aspergillus terreus | 1 (8 %) | |
| Fusarium solani | 3 (23 %) | |
| Fusarium species | 2 (15 %) | |
| Scedosporium apiospermum | 1 (8 %) | |
| Uniidentified dematiaceous fungus | 1 (8 %) | |
| Corneal button growth organism | Aspergillus flavus | 4 (31 %) |
| Aspergillus terreus | 1 (8 %) | |
| Scedosporium apiospermum | 1 (8 %) | |
| Uniidentified Hyaline fungus | 1 (8 %) | |
Table 3 outlines the donor corneal tissue characteristics. Donors had a mean age of 49 years (SD 20) at time of death with death to preservation time of 17.0 hours (SD 6.4) and a 7.5 days (SD 3.6). The mean cell count was 2300 cells/mm2 (SD 790). Intraoperatively, the mean donor graft size was large at 9.2 mm (SD 0.72). Intraoperatively, the patients were noted to have a hypopyon in 8 (62 %) and iris exudates in 7 (54 %).The cataractous lens was removed during surgery in 7/13 (54 %) cases with a vitrectomy performed in 4/13 (31 %) cases. The anterior chamber was noted to be fully formed at the end of the case in all patients. Intracameral voriconazole was administered in all cases (N = 13, 100 %).
Table 3.
Corneal donor characteristics.
| Overall (N = 13) | ||
|---|---|---|
| Age (years) | Mean (SD) | 49 (20) |
| Death to preservation time (hours) | Mean (SD) | 17 (6.4) |
| Death to surgery time (days) | Mean (SD) | 7.5 (3.6) |
| Average ECD (cells/mm2) | Mean (SD) | 2300 (790) |
| Corneal host size (mm) | Mean (SD) | 8.7 (0.66) |
| Corneal graft size (mm) | Mean (SD) | 9.2 (0.72) |
Fig. 1 shows examples of 6-month outcomes. At 6 months, 2/13 (15 %) patients had clear graft survival, mild corneal edema with good view to anterior chamber (AC) details and normal AC anatomy was noted in 4/13 (31 %), moderate to severe corneal edema with hazy view to the AC but normal AC anatomy was noted in 3/13 (23 %) and severe opacity with vascularization was noted in 4/13 (31 %). Of these, 9/13 (69 %) were determined to either have a good outcome or be a good candidate for optical PKP given lack of anterior segment abnormalities, severe glaucoma or corneal neovascularization. Thirty-one percent (4/13) were determined to have a poor prognosis for visual rehabilitation due to corneal neovascularization, secondary glaucoma, and/or retinal detachment.
Fig. 1.
6-Month Outcomes of Therapeutic Penetrating Keratoplasty for Fungal Keratitis Treated with Adjunctive Tacrolimus Ointment.
(A–F) Serial slitlamp photographs demonstrating the 6-month outcomes of patients who underwent therapeutic penetrating keratoplasty (TPK) for fungal keratitis and received adjunctive topical tacrolimus.
On average Best Spectacle Corrected Visual Acuity (BSCVA) improved by 3.5 logMAR lines (SD -0.06 to 0.77; P = 0.09). Mean BSCVA at 6 months was logMAR 1.9 (SD 0.69). Three patients had re-infection and required repeat TPK 3/13 (23 %). In these patients tacrolimus was stopped. There were no cases of endophthalmitis and no patients required urgent glaucoma surgery. Six month outcomes are summarized in Table 4.
Table 4.
6-Month outcomes.
| Overall (N = 13) | ||
|---|---|---|
| Graft Status | Clear graft | 2 (15 %) |
| Mild corneal edema | 4 (31 %) | |
| Moderate to severe corneal edema | 3 (23 %) | |
| Severe opacity with vascularization | 4 (31 %) | |
| BCVA (log MAR) | Mean (SD) | 1.9 (0.69) |
| Lens Status) | Aphakia | 8 (62 %) |
| Phakic | 2 (15 %) | |
| Pseudophakia | 3 (23 %) | |
| Repeat TPK | Yes | 2 (15 %) |
| Eligible for optical transplant | Clear graft, optical graft not required | 2 (15 %) |
| Good diagnosis for subsequent optical graft | 7 (54 %) | |
| Poor diagnosis for subsequent optical graft | 4 (31 %) | |
4. Discussion
In this series we observed graft failure, defined as irreversible moderate corneal edema or opacity in 54 % of TPKs for severe fungal keratitis by 6 months despite the addition of post-operative tacrolimus ointment. However, we also observed that anterior chamber anatomy was preserved without scarring in nearly 70 % of cases. This is an important outcome as it allows for successful visual rehabilitation with future optical penetrating keratoplasty (PKP). While 3 patients required repeat TPK for graft infection, there were no cases of endophthalmitis highlighting the safety of post operative tacrolimus in the setting of fungal keratitis. No patients required surgical interventions such as washing out the AC, re-suturing of the graft or glaucoma surgery. Another series from the same institution reported that 67 % of patients required subsequent procedures after TPK with 43 % developing endophthalmitis or secondary glaucoma.9 Therefore, the addition of tacrolimus did not seem to potentiate infection, and may have reduced inflammation thereby minimizing other complications.
Although TPK may be successful at eliminating the infection, graft survival in the setting of active infection and inflammation is poor.10, 11, 12 In one study, approximately 51 % of TPK grafts had failed by 6 months, increasing to 70 % at 1 year.13 During acute infection, pathogens, keratocytes and other inflammatory cells secrete enzymes that promote protein degradation and keratolysis. Controlling the host inflammatory response may be beneficial, however, due to concerns for recurrent infection, topical steroids are not started in the post-operative period. This inflammation leads to an increased risk of graft rejection and failure, as well as scarring of the iris and anterior chamber angle, which reduces the chance of success for a subsequent optical keratoplasty.
Tacrolimus (FK506) is a novel therapeutic in the treatment of fungal keratitis which has the potential to address both infection and inflammation in fungal keratitis requiring TPK. Inhibition of the Calcineurin stress response pathway in fungi may improve outcomes in fungal keratitis requiring TPK. In vitro testing has shown that tacrolimus has broad inhibitory effect on common ocular pathogens such as Aspergillus and Fusariums spp and may have a synergistic effect with common antifungals such as caspofungin and voriconazole.3,14 In a mouse model of fungal keratitis, those treated with both natamycin and FK506 showed reduced fungi-induced corneal damage on histopathology compared to those treated with either drug alone, possibly through inhibition of fungi-induced activation of the NLRP3 inflammasome.15
Tacrolimus has also been shown to reduce graft rejection both in rat PKP models and in several human studies.16, 17, 18 A randomized clinical trial of high-risk penetrating keratoplasty patients found a statistically significant reduced graft rejection to 16 % among those treated with topical FK506 0.1 % compared with 46 % among those treated with topical Cyclosporine 1 %.16,19,20 Among heart and liver transplant patients, the rate of invasive aspergillosis and mortality has been found to be much reduced in those treated with oral calcineurin inhibitors.21
FK506 is unique in its potential to address both the infection and inflammation simultaneously. Adjunctive topical tacrolimus after therapeutic penetrating keratoplasty may reduce post operative complications and improve the chances of subsequent successful optical penetrating keratoplasty. Improving outcomes of severe fungal keratitis is of utmost importance to the practicing corneal specialists who manage these conditions. Limitations to this study include the fact that it is a small prospective series. Additionally, patients received topical steroids at one month post-operatively in this study which may make it difficult to discern the benefit of topical tacrolimus. The first month following TPK is critical for managing inflammation in fungal keratitis. As steroids are contraindicated, the use of adjunctive tacrolimus during this period was studied and found to have potential benefits. A larger randomized clinical trial is necessary to fully evaluate the benefit of the topical adjunctive tacrolimus.
CRediT authorship contribution statement
S. Josephine Christy: Writing – review & editing, Writing – original draft, Investigation, Data curation, Conceptualization. Kunal Mandlik: Methodology, Investigation. Vasantha Kumar: Methodology, Investigation. Srinivas Sai Suma Maalika Kanchugantla: Writing – review & editing, Validation, Data curation. Sarah Abdelrahman: Validation, Investigation, Data curation, Conceptualization. Jennifer Rose-Nussbaumer: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization.
Disclosure
None of the authors have a proprietary/financial interest to disclose.
Ethics
Ethical approval was obtained from the University of California, San Francisco, Committee on Human Research (18–26045), Stanford University Committee on Human Research, The Aravind Eye Care System Institutional Review Board, and Pondicherry (AEH/PDY/EC/OA/85/2020), India; Approval was also obtained from The Indian Council of Medical Research (ICMR: 2019–4148). Written informed consent was obtained from all participants, and the trial conformed to the Declaration of Helsinki. The Data and Safety Monitoring Committee performed regular reviews for safety, data quality, and trial conduct.
Contribution statement
J Rose-Nussbaumer, Josephine Christy, Kunal Mandlik and Vasantha Kumar have contributed to the design of the study and study implementation and these collaborators make up the steering committee. Sarah Abdelrahman and Srinivas Sai Suma Maalika Kanchugantla are contributing to study implementation.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by grants UG1 EY028518 (Lietman/Rose-Nussbaumer) and K23 EY025025 (Rose-Nussbaumer) from the National Eye Institute P30 grant from the National Eye Institute (Stanford) and Research to Prevent Blindness (UCSF & Stanford University). Our study team would like to thank Dr. Rajesh Betdur from Aravind Eye Hospital, Pondicherry, India for providing his expertise on the study.
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