Photoactivation holds promise for keratitis treatment

      The same photoactivation process used in collagen cross-linking for keratoconus can kill bacteria without the need for the oxygen responsible for the biomechanical effects, potentially pointing toward better treatments for keratitis, according to Olivier Richoz, MD, PhD, a corneal and anterior segment fellow at Belfast Royal Victory Hospital, Belfast, United Kingdom.  

Related: Addressing the challenges of Acanthamoeba keratitis

All images courtesy of Olivier Richoz, MD, PhD.

To read the full article without accompanying images, click here. 

When using collagen cross-linking to treat keratoconus, clinicians anesthetize the patient’s cornea, apply 0.1% riboflavin, and expose the cornea to 365 nm ultraviolet light. Free radicals created by this irradiation create new covalent bonds between collagen fibers, which strengthens the cornea.

More: When is crosslinking appropriate?

The treatment also kills pathogens and has been used in infectious keratitis since 2008, Dr. Richoz said.

Recent: Predicting, treating keratoconus in 2016

Some pathogens causing the disease, such as Fusarium solani, can be resistant to common anti-fungal medications.

Related: Why infections related to PK require intense vigilance

About 60,000 people in the United States and 2 million people in India get keratitis every year, he said. 

More: What literature review says about modern LASIK

Having shown in previous research that the biomechanical effects of collagen cross-linking depend on oxygen, Dr. Richoz wanted to know whether the same was true of the anti-bacterial effects of the treatment.

The question proved difficult to answer because photoactivation has a strong effect on only the first 100-150 µm of the cornea. “That means if you want to analyze the killing rate of bacteria, you need to create an experimental setting that uses extremely small slides of cornea,” Dr. Richoz said.

After a year of experimenting, he created conditions in which the hypothesis could be tested. After incubating the Staphylococcus aureus and Pseudomonas aeruginosa with riboflavin for 30 minutes, he put the bacteria on discs of cornea 150 µm in thickness and 10 mm in diameter.

He treated some of the cornea discs for 5 minutes at 18 mW/cm² of ultraviolet light in the presence of oxygen, and others in an oxygen-free environment. He did not treat some of the cornea discs at all. Then he put the cornea discs in a 0.9% solution of sodium chloride for 60 minutes.

Next, he plated the solution on Mueller-Hinton agar, incubated it for 24 hours, and counted the number of colony-forming units.

Compared to the control discs, the irradiated discs had only 1% of the S. aureus and 2% of the P. aeruginosa when the irradiation took place in the presence of oxygen. In the oxygen-free environment, 5% of the S. aureus and 50% of the P. aeruginosa survived.

From this, Dr. Richoz concluded that free radicals are only partially responsible for the death of bacteria when riboflavin is photoactivated. He theorizes that the interaction of riboflavin with DNA in the bacteria may be a more important effect.

“We know that the riboflavin can react with the DNA,” he said. “But we don’t know if the killing rate is only due to the effect of the riboflavin on the DNA, or maybe the riboflavin can interact with something else in the bacteria.”

If the process can work with riboflavin, perhaps it will work with other molecules such as antibiotics. “The idea is to choose a molecule in the future that is specific to the bacteria and to photoactivate that molecule,” he said.

This would avoid the risk of damage to the patient’s healthy tissue. He has already used software to predict the reactivity of molecules in various antibiotics.

Damage to healthy cornea from photoactivated riboflavin is not a significant risk because stromal cells regenerate within a year of treatment, Dr. Richoz said.

But making the photoactivation more selective to pathogens could be important in other types of infections or in cancer, he said. Dr. Richoz has conducted small experiments in animals with skin infections caused by antibiotic-resistant bacteria. In these experiments, he used blue light rather than ultraviolet light since blue light is non-ionizing and does not damage healthy skin cells.

The experiments were successful. “If we inject the antibiotics to which the bacteria is resistant, and we subject the bacteria to a specific pattern of blue light, the bacteria becomes sensitive again,” Dr. Richoz said. “It was very exciting.”

Dr. Richoz has a patent on this technology and also on the use of slit lamps which could be used to inexpensively to generate the necessary radiation, an approach that could be particularly useful in developing countries where more sophisticated equipment is not available, he said.

For now, he is focusing on the use of scleral cross-linking, which he believes could be used to treat progressive myopia.

“The idea is to use a similar technology as corneal cross-linking, but in this case to cross-link the sclera in the back of the eye,” he said. “It’s extremely difficult. You need a miniaturized device to access the posterior part of the eye.”

Full article

The same photoactivation process used in collagen cross-linking for keratoconus can kill bacteria without the need for the oxygen responsible for the biomechanical effects, potentially pointing toward better treatments for keratitis, according to Olivier Richoz, MD, PhD.

Dr. Richoz, a corneal and anterior segment fellow at Belfast Royal Victory Hospital, Belfast, United Kingdom, presented the finding at the 2015 American Academy of Ophthalmology (AAO) meeting.

When using collagen cross-linking to treat keratoconus, clinicians anesthetize the patient’s cornea, apply 0.1% riboflavin, and expose the cornea to 365 nm ultraviolet light. Free radicals created by this irradiation create new covalent bonds between collagen fibers, which strengthens the cornea.

The treatment also kills pathogens and has been used in infectious keratitis since 2008, Dr. Richoz said. Some pathogens causing the disease, such as Fusarium solani, can be resistant to common anti-fungal medications.

About 60,000 people in the United States and 2 million people in India get keratitis every year, he said. 

Having shown in previous research that the biomechanical effects of collagen cross-linking depend on oxygen, Dr. Richoz wanted to know whether the same was true of the anti-bacterial effects of the treatment.

The question proved difficult to answer because photoactivation has a strong effect on only the first 100-150 µm of the cornea. “That means if you want to analyze the killing rate of bacteria, you need to create an experimental setting that uses extremely small slides of cornea,” Dr. Richoz said.

After a year of experimenting, he created conditions in which the hypothesis could be tested. After incubating the Staphylococcus aureus and Pseudomonas aeruginosa with riboflavin for 30 minutes, he put the bacteria on discs of cornea 150 µm in thickness and 10 mm in diameter.

He treated some of the cornea discs for 5 minutes at 18 mW/cm² of ultraviolet light in the presence of oxygen, and others in an oxygen-free environment. He did not treat some of the cornea discs at all. Then he put the cornea discs in a 0.9% solution of sodium chloride for 60 minutes.

Next, he plated the solution on Mueller-Hinton agar, incubated it for 24 hours, and counted the number of colony-forming units.

How much bacteria survived?

Compared to the control discs, the irradiated discs had only 1% of the S. aureus and 2% of the P. aeruginosa when the irradiation took place in the presence of oxygen. In the oxygen-free environment, 5% of the S. aureus and 50% of the P. aeruginosa survived.

From this, Dr. Richoz concluded that free radicals are only partially responsible for the death of bacteria when riboflavin is photoactivated. He theorizes that the interaction of riboflavin with DNA in the bacteria may be a more important effect.

“We know that the riboflavin can react with the DNA,” he said. “But we don’t know if the killing rate is only due to the effect of the riboflavin on the DNA, or maybe the riboflavin can interact with something else in the bacteria.”

If the process can work with riboflavin, perhaps it will work with other molecules such as antibiotics. “The idea is to choose a molecule in the future that is specific to the bacteria and to photoactivate that molecule,” he said.

This would avoid the risk of damage to the patient’s healthy tissue. He has already used software to predict the reactivity of molecules in various antibiotics.

Damage to healthy cornea from photoactivated riboflavin is not a significant risk because stromal cells regenerate within a year of treatment, Dr. Richoz said.

But making the photoactivation more selective to pathogens could be important in other types of infections or in cancer, he said. Dr. Richoz has conducted small experiments in animals with skin infections caused by antibiotic-resistant bacteria. In these experiments, he used blue light rather than ultraviolet light since blue light is non-ionizing and does not damage healthy skin cells.

The experiments were successful. “If we inject the antibiotics to which the bacteria is resistant, and we subject the bacteria to a specific pattern of blue light, the bacteria becomes sensitive again,” Dr. Richoz said. “It was very exciting.”

Dr. Richoz has a patent on this technology and also on the use of slit lamps which could be used to inexpensively to generate the necessary radiation, an approach that could be particularly useful in developing countries where more sophisticated equipment is not available, he said.

For now, he is focusing on the use of scleral cross-linking, which he believes could be used to treat progressive myopia.

“The idea is to use a similar technology as corneal cross-linking, but in this case to cross-link the sclera in the back of the eye,” he said. “It’s extremely difficult. You need a miniaturized device to access the posterior part of the eye.”