1. Crosslinking for Microbial Keratitis To Treat or not to Treat?
Dr. Kendrick Shih MBBS, MRes (Medicine) MRCSEd, FCOphthHK, FHKAM
Department of Ophthalmology
Li Ka Shing Faculty of Medicine

2. Acknowledgements Professor Vishal Jhanji, UMPC Eye Centre, USA
Dr. Tommy Chan, DOVS The Chinese University of Hong kong, Hong Kong SAR

3. Declaration
I do not have any financial interests in the drugs or products discussed in this presentation

4. Introduction
The current mainstay of microbial keratitis treatment is the use of local antibiotics
However the treatment takes time to for effect (time is cornea) and is associated with potentially serious ocular surface toxicity and microbial resistance
Cornea cross-linking (CXL) is an established treatment for preventing progressive keratoconus or ectasia
Increases biomechanical strength of the anterior cornea stroma
Since 2000, the use of CXL has been extended to the treatment of microbial keratitis as form of adjunctive treatment – Photoactivated Chromophores for Cross-linking (PACK-CXL)
Randleman JB et al. Surv Ophthalmol. 2015
Schnitzler E et al. Klin Monbl Augmenheikd. 2000
Iseli HP et al. Cornea. 2008

5. Mechanism of action: Not Fully Known
Photo-activation of a chromophore can release reactive oxygen species and theoretically reduce microbial load, similar to a disinfectant.
Oxidation of pathogen DNA and RNA
ROS damage of pathogen cell walls
Increased corneal stiffness and resistance to enzymatic digestion
Killing of inflammatory and immune cells (apoptosis)
Kumar V et al. Photochem Photobiol. 2004
Martins SA et al. IOVS. 2008
Spoerl E et al. Curr Eye Res. 2004
Wollensak G et al. Cornea. 2004

6. PACK-CXL Protocol Standard Dresden protocol most commonly used
Epithelium-off
Application of isotonic riboflavin for 30 minutes (5 minute intervals)
365-nm UVA light exposure (3mW/cm2) for 30 minutes
Anterior segment optical coherence tomography (AS-OCT) is a useful diagnostic tool for assessing infiltrate depth before treatment and monitoring afterward
A minimal corneal thickness of 400 µm is required to prevent any endothelial damage from CXL

7. Important Considerations
Intraoperative pachymetry and hypotonic riboflavin may help ensure sufficient endothelial protection during CXL in borderline thin corneas.
Use of riboflavin/UV-A should be avoided in patients with a history or suspicion of herpes simplex. Herpes is activated by ultraviolet irradiation
Fluorescein stain should be avoided before use of photo-activated riboflavin, because it competes for absorption of UV-A light and reduces antimicrobial efficacy
The total number of reported acanthamoeba cases treated with PACK-CXL is too few to accurately assess success rate and confidence interval. Given the mixed results with cysts and trophozoites in laboratory studies and that the infectious organisms are often deep in the cornea after prolonged infection,

8. Proposed Variations to Standard Dresden Protocol
Important to note that Dresden protocol was not designed to target microbial keratitis:
Avoid enlarging epithelial defect to avoid delayed healing vs
Remove epithelium beyond area of observed infiltrate to improve riboflavin penetration
Alter riboflavin concentration to improve killing effect
Higher concentration (0.25 vs 0.1%) for ↑ fungicidal effect
Lower concentrations (0.03 vs 0.09% with longer UVA exposure) for ↑ bactericidal effect
Prolonging riboflavin instillation time ↑ collagen compaction, ↑ resistance to enzymatic digestion
May use accelerated protocol (16 mW/cm2 for 5 min, 36 mW/cm2 for 2.5 min) – controversial

9. Major Questions How safe and effective is PACK-CXL?
Adjuvant therapy vs monotherapy?
What type of microbial keratitis can be treated with PACK-CXL?
When is the optimum time point for treating microbial keratitis with PACK-CXL?
The total number of reported acanthamoeba cases treated with PACK-CXL is too few to accurately assess success rate and confidence interval. Given the mixed results with cysts and trophozoites in laboratory studies and that the infectious organisms are often deep in the cornea after prolonged infection,

10. What Does the Evidence Say
Approximately 30 published studies to date:
2 systematic reviews and 1 meta-analysis
2 randomized controlled trials
15 case series
10 case reports
Treatment for bacteria, fungal species, acanthamoeba cysts , trophozoites
Most employed standard Dresden protocol
Major limitations include lack of masking, a heterogenous group of conditions with different microbial agents and differences in patient demographics
Price MO et al. Curr Opin Ophthalmol. 2016
Chan TC et al. Curr Opin Ophthalmol. 2016

11. How Safe and Effective is PACK-CXL?
Overall 87% success rate with PACK-CXL for microbial keratitis (defined as complete re-epithelialization and infiltrate resolution)
Similar to topical antibiotic therapy (5-year historical comparison and control groups in RCTs), although in one RCT PACK-CXL group had patients with larger ulcers
HOWEVER, may significantly shorten re-epithelialization in early cases (17 vs 24 days)
No difference in re-epithelialization rate and final VA in advanced cases, but treated group had no recurrences or perforations
Papaioannou et al. Cornea. 2016
Said et al. Ophthalmology. 2014
Bamdad et al. Cornea. 2015
Price et al. J Refract Surg. 2012
Vajpayee et al. Clin Experiment Ophthalmol. 2015

12. How Safe and Effective is PACK-CXL?
Additional observations:
May have role in antibiotic-resistant cases
Smaller ulcers associated with higher success rates in bacterial and fungal keratitis
Presence of hypopion associated with lower success rate

13. Adjuvant Therapy vs Monotherapy?
Vast majority of studies used PACK-CXL as adjuvant therapy
Only 3 studies had patients with use of PACK-CXL as monotherapy
Makadoumi et al had 16 patients with bacterial keratitis (unknown if consecutive or not) who all had re-epithelialization and resolution of infiltrate with PACK-CXL alone
Tabibian et al had one case of fungal keratitis treated with accelerated PACK-CXL (9 mW/cm2 for 10 min) that healed within 3 days
Fisher et al had one case of fungal keratitis treated with standard PACK-CXL that healed within 3 days as well
Makdoumi et al. Graefes Arch Clin Exp Ophthalmol. 2012
Tabibian et al. J Refract Surg. 2014
Price et al. J Refract Surg. 2012

14. Which Microbes are Most Susceptible?
Mixed results for heterogenous groups of microbial keratitis from largest RCT
Faster recovery in bacterial keratitis with infiltrate size less than 2.5 mm
Poor response to CXL in fungal keratitis
Inadequate case number to accurately assess success rate for acanthamoeba keratitis
Price et al. J Refract Surg. 2012

15. Optimum Treatment Time Point?
A better treatment response with superficial corneal infiltrate involving the anterior third of the stroma
However debatable whether depth of infection should be monitored via ASOCT (infiltrate depth) vs confocal microscopy (microbial depth), especially for deeply penetrating conditions like fungal keratitis
Deeper microbial penetration associated with disease flares after PACK-CXL
Alsherhri et al demonstrated in ex vivo human corneas that PACK-CXL effectively reduced microbial load up to treatment penetration depth on confocal microscopy
Shetty et al. Br J Ophthalmol. 2014
Price et al. J Refract Surg. 2012
Alshehri et al. IOVS. 2016

16. Summary
Cornea collagen cross-linking is a potentially useful adjunctive treatment for the management of selective cases of bacterial keratitis
Existing evidence does not support its routine use in fungal or amoebic keratitis
Treatment effectiveness is limited by depth of cross-linking ( μm). Therefore it may be potentially more useful in early keratitis
Richoz et al have recently shown that an accelerated protocol using 18 mW/cm2 for 5 minutes and even 36 mW/cm2 for 2.5 minutes allows to maintain the same high bacterial killing rate observed in earlier studies using the Dresden protocol

17. Future Prospect
Larger randomized, controlled studies are needed to accurately assess the safety and efficacy of optimized PACK-CXL protocols relative to standard antimicrobial treatments
Improving the treatment by modifying parameters such as time, duration of irradiance and type of chromophore
Richoz et al have recently shown that an accelerated protocol using 18 mW/cm2 for 5 minutes and even 36 mW/cm2 for 2.5 minutes allows to maintain the same high bacterial killing rate observed in earlier studies using the Dresden protocol

18. Thank You