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Ultraviolet Light A (UVA) Photoactivation of Riboflavin as a Potential Therapy for Infectious Keratitis

“If I had fifty-three minutes to spend, I would walk very slowly towards a spring of fresh water …” -Antoine de Saint-Exupéry, The Little Prince

Örebro Studies in Medicine 63

KARIM MAKDOUMI

Ultraviolet Light A (UVA) Photoactivation of Riboflavin as a Potential Therapy for Infectious Keratitis

© Karim Makdoumi, 2011

Title: Ultraviolet Light A (UVA) Photoactivation of Riboflavin as a Potential Therapy for Infectious Keratitis.

Publisher: Örebro University 2011

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Print: Ineko, Kållered 11/2011

ISSN 1652-4063 ISBN 978-91-7668-834-2

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Abstract Karim Makdoumi (2011): Ultraviolet Light A (UVA) Photoactivation of Riboflavin as a Potential Therapy for Infectious Keratitis. Örebro Studies in Medicine 63, 70pp. Collagen Crosslinking (CXL) is a treatment based on the photosensitization of riboflavin (vitamin B2), using ultraviolet light (UVA). It is implemented as an alternative to transplantation in keratoconus and corneal ectasia. The same mechanism is utilized in transfusion medicine, to reduce the risks for infectious transmission associated with the procedure. Infectious keratitis is a condition that is coupled with risks for the development of serious complications and subsequent visual impairment or even blindness. As the spread of antibiotic resistant bacteria signifies that real hazard and concern that corneal infection in the future could be a severely difficult condition to treat, the need for new therapeutics seems evident.

The aim of this thesis was to study the photoactivation of riboflavin and to elucidate several key factors involved in the antimicrobial action of the phenomenon as well as to study the clinical effect of CXL in infectious keratitis.

The experimental papers investigated the antimicrobial effect tested on three different bacterial strains, commonly found as causative microorganisms in keratitis, as well as Acanthamoeba castellanii, in fluid solutions. The purpose was to establish if UVA alone eliminated microbes or whether the outcome was mediated by the combined action with riboflavin and if so, try to specify the quantity required for achieving the effect. A clear bactericidal effect was seen in all tested strains, with results strongly indicating an interaction between the vitamin and ultraviolet light. Regarding Acanthamoeba however, growth inhibition was induced by ultraviolet light solitarily, with no additional effect from riboflavin.

The clinical response of riboflavin photoactivation, employed as CXL was observed in 7 severe cases of infectious keratitis, which all responded to therapy. A clinical non-randomized pilot study of UVA-riboflavin interaction as the primary therapy for bacterial keratitis resulted in curing of 14 out of 16 ulcers without the use of antibiotics.

In conclusion, the use of UVA photoactivation of riboflavin as an infectious photodynamic therapy seems to be a promising tool for integration as an adjuvant treatment in infectious keratitis.

Keywords: riboflavin; ultraviolet; light; UV; UVA; keratitis; melting; bacteria; acanthamoeba; photoactivation; photosensitization Karim Makdoumi, Hälsoakademin Örebro University, SE-701 82 Örebro, Sweden, [email protected]

CONTENTS

ABBREVIATIONS .................................................................................... 9

INTRODUCTION .................................................................................. 11 Corneal Collagen Crosslinking (CXL) ...................................................... 11 Techniques ............................................................................................... 12 Safety of CXL .......................................................................................... 14 Riboflavin and its Photoactivation ........................................................... 15 Riboflavin in Pathogen Eradication Technology (PET)/Pathogen Reduction Technology (PRT) .................................................................................... 17 Microbial Keratitis ................................................................................... 18 Antibiotic Resistance ................................................................................ 21 Structure of the Cornea ............................................................................ 24 Corneal Immunology ............................................................................... 26 CXL in Keratitis ....................................................................................... 27 Experimental Investigations ..................................................................... 27 Clinical Results ........................................................................................ 27

AIMS ....................................................................................................... 29

PATIENTS ............................................................................................... 31

MATERIALS AND METHODS .............................................................. 33

RESULTS AND DISCUSSION ................................................................ 37

CONCLUSIONS ...................................................................................... 47

POPULÄRVETENSKAPLIG SAMMANFATTNING ............................. 49

ACKNOWLEDGEMENTS ...................................................................... 53

REFERENCES ......................................................................................... 55

KARIM MAKDOUMI Riboflavin Photoactivation & Keratitis 9

Abbreviations BCL – Bandage Contact Lens CFU – Colony Forming Units CXL – Corneal Collagen Crosslinking FAD – Flavin Adenine Dinucleotide FDA – United States Food and Drug Administration FMN – Flavin Mononucleotide GAG – Glucose-amino-glycan GRAS – Generally Recognized As Safe GVHD – Graft-versus-Host Disease ICRS – Intra-stromal Corneal Ring Segment LASIK – Laser Assisted in SItu Keratomileusis MMP – Matrix Metallo Proteinase PET – Pathogen Eradication Therapy PRK – Photorefractive Keratectomy PRT – Pathogen Reduction Technology ROS – Reactive Oxygen Species SEM – Scanning Electron Microscope UVA – Ultraviolet Light A WBC – White Blood Cell


Introduction

Corneal Collagen Crosslinking (CXL)

In 2003 the first clinical results were published regarding a novel therapy for keratoconus, using Corneal Collagen Crosslinking or CXL 1, 2. The aim of this treatment is to augment the biomechanical strength of the corneal stroma, hence targeting the inherent weakness of the tissue, which is considered as the main dilemma associated with the condition 3. Promising results regarding the arrest of disease progression have been published by several groups world-wide, with data involving results for both keratoconus and post-LASIK ectasia either as a single therapy 4-10 or in combination with other treatment modalities, such as PRK and intra-stromal corneal ring segments (ICRS) 11-15.

A research group at the Dresden Technical University developed the CXL technology in the 1990s 2 by proposing the concept of utilizing riboflavin excitation through photoactivation by Ultraviolet Light A (UVA). This results in the creation of reactive oxygen species (ROS) which mediate a biological polymerization by the production of new covalent bonds between collagen molecules in the corneal stroma, increasing its stiffness 16-18. Consequently, the biomechanical resistance in the human cornea is increased by approximately 330 % under in vitro conditions 19. The procedure also results in changes which elevate the tissue thermal shrinkage temperature 20 and makes the cornea more resistant to the action of several collagen degrading enzymes 21. As the wavelength of the ultraviolet light has been selected to coincide with the 370 nm peak of the riboflavin absorption spectrum, as well as to minimize the passage of UVA through the entire corneal thickness, the effect of CXL is mainly limited to the anterior 300 μm of the stroma 22-24. Riboflavin instillation reduces the penetration of light further, leading to a transmission of approximately seven percent passing the cornea. Hence, cell damages at the endothelial level should not occur and the cytotoxic threshold values regarding lens and retina are not reached using the standard protocol 3. Intraoperative pachymetry is strongly recommended when conducting the procedure, to minimize the risk for endothelial damage by avoiding hazardous UV dosages 25, 26. In the anterior part of the corneal stroma, apoptosis of keratocytes can be observed, followed by a repopulation of novel cells 24, 27-29. Clinical use of the method has not indicated that it is associated with endothelial cell damage 28, 29.

12 KARIM MAKDOUMI Riboflavin Photoactivation & Keratitis

Figure 1: Illustration mechanisms of CXL, by formation of new bonds between collagen molecules. (Image courtesy of Professor Michael Mrochen, Institute for Refractive and Ophthalmologic Surgery (IROC), Zürich, Switzerland)

Techniques

As a rule the surgical procedure is performed using local anesthesia. Initially, a corneal abrasion is done, either with a blunt instrument or wiping the epithelium off with a sterile swab after brief ethanol application. Pachymetry should be carried out to determine that the corneal thickness after epithelial removal exceeds 400 µm, which is required to ascertain safety regarding the endothelial cells. Under standard conditions topical administration of 0.1 % isotonic riboflavin preparation (10 mg riboflavin-5-phosphate in 10 ml dextran 20 % solution) is carried out for 30 minutes, given at minimum every 5 minutes, followed by slit-lamp examination to assess if sufficient uptake of the vitamin solution has occurred. This is confirmed by the observation of a slightly yellow-colored cornea and a flare in the anterior chamber. If such signs are absent the instillation of riboflavin has to be continued until they can be adequately detected. Subsequently, illumination


takes place for another 30 minutes, at a standardized irradiance (3 mW/cm2) and ultraviolet light dose (5.4 J/cm2) 1, 2. In thin corneas, explicitly with a corneal thickness below 400 µm after epithelial removal upon measurement with pachymetry, the customary riboflavin solution used can be substituted by a hypotonic preparation with the same vitamin B2 concentration. Osmotic action through the administration of this solution can in selected cases swell the cornea up to the generally accepted threshold, thus allowing UVA illumination without jeopardizing the endothelium 30. If the iatrogenic corneal edema is insufficient the ultraviolet light exposure cannot take place and the operation must be discontinued.

In view of the fact that epithelial removal in general causes severe patient discomfort postoperatively, it has been appealing to implement the surgical technique through a trans-epithelial approach (a technique given the abbreviation C3-R). Different methods to achieve this have been proposed, such as topical anesthetic drops or benzalkonium chloride administration to loosen tight junctions in the epithelium combined with riboflavin (0.1 %) in a carboxymethylcellulose preparation without dextran 31. The UV passage without a complete epithelial removal is noticeably reduced why the crosslinking effect is significantly lower than compared to the standard practice 32-35. Nevertheless, the transepithelial procedure has been suggested as a potential choice of therapy in exceedingly thin corneas, where the swelling through hypotonic solutions is not satisfactory 35.


Figure 2: Clinical treatment of keratoconus using the CXL method. The picture shows the illumination phase of the procedure.

Safety of CXL

In Europe the procedure has been accepted in the routine management of keratoconus and other corneal ectasia whereas it is not presently approved for use in the USA. The U.S. Food and Drug Administration (FDA) phase III clinical trials have been launched and some results regarding outcome variables have been published 5, 36. Postoperative side effects along with treatment complications are rare and the most commonly described are corneal haze, which in the majority of cases is temporary 37-39, corneal melting in rare cases 40-42, and there are several accounts of subjects who developed microbial keratitis. In most of these patients a bandage contact lens (BCL) had been applied after the CXL and in several of them poor hygiene control associated with lens wear was described 43-46. Activation of herpetic keratitis with iritis shortly after the procedure 47 has been reported


in one patient and a description of a case with diffuse lamellar keratititis post-CXL, in an eye with post-LASIK ectasia 48. Follow-up after UVA-riboflavin crosslinking for keratoconus, including examination with in vivo confocal microscopy, has demonstrated that neither limbal nor endothelial cell counts or configuration are noticeably negatively influenced by the treatment. The apoptosis of keratocytes can be detected to a depth of 340 microns at the most and disappearance of nerve-fibers occurs to the middle of the stroma. Regeneration of the mentioned alterations is initiated within the first two months and seems to be completed within half a year 28, 49-52. After CXL a higher density of stromal collagen fibers has been confirmed by in vivo confocal microscopy 49, 51, indicative of the maintained crosslinking effect. No alterations regarding the foveal structure have been noted on Optical Coherence Tomography (OCT) examinations 50. Anterior segment OCT, however, has displayed a stromal demarcation line signifying the limit of the stabilizing effect generated by the procedure 29 and total corneal aberrations are evidently lower at follow-up on aberrometric measurement 53, 54. The use of CXL is concordantly regarded as a safe procedure with low complication rates, if practised according to recommendations 50, 55-58.

Riboflavin and its Photoactivation

Riboflavin, or vitamin B2, plays an important role in several metabolic processes, particularly energy metabolism. It is essential for the coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), which act as electron carriers. Naturally occurring in foods such as green leafy vegetables, meats, milk, and dairy products 59, the vitamin is also, due to its yellow color, used as a food dye agent under the name E101 60. In its oxidized state it has light absorption peaks in ultraviolet range around wavelengths 221-227, 265-270, 365-370 nm, and in visible light spectrum in the region of 445 nm 61, meaning that irradiation using either of these leads to excitation of riboflavin, followed by its degradation.


Figure 3: Absorption spectrum of riboflavin in water. (Courtesy of Professor Michael Greenlief, Director, Charles W. Gehrke Proteomics Center and the MU NMR Facility University of Missouri-Columbia, USA) Excitation leads to fluorescence in the green light spectrum at a wavelength of 520-560 nm and after illumination the vitamin is bleached 62. At normal and acidic pH riboflavin subjected to light primarily degrades into lumichrome (at alkaline pH the principal photodegradation product is lumiflavin). Less abundant products are 2’-keto-flavin, 4’-keto-flavin, and formylmethylflavin 63, all of which naturally occur in the human body 64. The photosensitization of riboflavin involves generation of ROS, principally by production of singlet oxygen 65, 66, a central intermediate for the cytotoxic action in photodynamic therapy (PDT) for treatment of cancer and age related macular degeneration 67-69. Also, superoxide anion radical (O2

-), the riboflavin radical as well as hydrogen peroxide (H2O2) can be produced by the illumination of the vitamin with UVA 70-73. H2O2 has considerably longer half-life than singlet oxygen and diffuses freely past cellular membranes, why it is believed to be of main importance in the cytotoxicity of vitamin B2 excitation 72, 73.

Lysis of riboflavin through illumination and the associated production of ROS cause changes in the environmental biological structures and already in 1965 it was first described that the process can cause RNA inactivation in the tobacco mosaic virus 74. DNA is furthermore oxidized by the phenomenon, detectable by the production of 8-hydroxydeoxyguanosine 75 and riboflavin has been targeted as one of the molecules possibly involved in skin aging 73, 76 along with other biological effects 77.


Nonetheless, the toxicity properties of vitamin B2 along with its degradation compounds are extremely favourable and riboflavin has been labelled as a substance generally recognized as safe (GRAS) by the U.S. FDA 78. There is no evidence that limited illumination of this molecule has carcinogenic or mutagenic effects, which has been supported by long term follow-up of a large number of children, showing no increased tendencies to develop cancer after UV illumination of neonates for icterus 79, a process known to involve photodegeneration of riboflavin 80-82.

Riboflavin in Pathogen Eradication Technology (PET)/Pathogen Reduction Technology (PRT)

The ability of riboflavin excitation by ultraviolet light to induce RNA/DNA damage of pathogens is a phenomenon that has been extensively studied and it can achieve an efficient eradication of several viruses, bacteria and parasites 78, 83-87. Microbial elimination is believed to be mediated partly by non-specific ROS damage but intensified by the intercalation of the planar part of riboflavin between base pairs in the DNA and RNA of pathogens, thus inducing strand cleavage through guanine oxidation 78. This antimicrobial efficacy has consequently led to the development of a commercially available medical technical device to increase the safety of transfusions, through inactivation of micro-organisms by way of riboflavin UV irradiation 88-91. Figure 4: Intercalation of riboflavin, mediating the antimicrobial effect utilized in PET/PRT. (Reprinted from Bryant BJ, Klein HG. Pathogen inactivation: the definitive safeguard for the blood supply. Arch Pathol Lab Med. 2007 May;131(5):719-33. Permission from Archives of Pathology & Laboratory Medicine. Copyright 2007. College of American Pathologists.)


Pathogen Eradication Technology and Pathogen Reduction Technology are collective terms for the different methods used for eliminating possible microbial contaminants in transfusions. Since only limited testing for possible pathogens is conducted in donors of blood products, such as HIV and hepatitis, there exists a potential risk for transmittal of infectious diseases during a transfusion. By UV illumination of different compounds pathogens can be effectively inactivated, thus decreasing the risk for recipients acquiring infections. Molecules of interest for this purpose are methylene blue, psoralens (in particular S-59 or amotosalen), riboflavin, and other compounds 92-94. Methylene blue is applied in clinical practice regarding plasma transfusions via THERAFLEX MB-Plasma® 95, 96 and two other devices are CE certified in Europe, for the treatment of platelet transfusions, in order to increase safety for recipients, sold under the names INTERCEPT Blood SystemTM (S-59) 97-99 and Mirasol® PRT system (riboflavin) 86, 100-102. The UV light exposure doses in Mirasol® and collagen crosslinking are similar, however in the prior a spectrum between 220 and 370 nm is used, whereas in CXL only monochromatic light of 365 or 370 nm has been implemented for illumination. Aside from the antimicrobial effect mediated by photochemical interaction by UV and riboflavin, the process has an efficacious capacity to inactivate white blood cells (WBC) 103-

105, which has led to the raised interest for the possible prevention of transfusion associated Graft-versus-Host disease (GVHD) through the photooxidative procedure 106, 107.

Microbial Keratitis

A Corneal Infection, or Microbial Keratitis, is a condition that often involves sudden as well as intense symptoms, including a pericorneal or mixed injection, pain, and acute visual loss. The associated inflammation can be very pronounced and the condition needs to be forcefully addressed in order to prevent disease progression, reduce complication rate, and limit vision loss. Albeit relatively rare, the condition is associated with a risk for severe deterioration of visual acuity 108, 109 and even blindness 110, 111. Corneal ulceration with subsequent scarring is an important causative factor for loss of vision and constitutes a fraction of global blindness 112, 113. The most frequently reported risk factors isolated for corneal infections involve contact lens wear, ocular trauma, ocular surface disease, herpetic keratitis, multifactorial genesis, prior ophthalmic surgery, and systemic diseases (such as diabetes mellitus, rheumathoid arthritis, and immune deficiency syndromes) 108-111, 114.


Figure 5: An example of Microbial Keratitis with associated intense inflammatory response. As contact lens use and ocular trauma are more common at younger ages and ocular surgery largely involves the elderly population these risk factors tend to dominate each category respectively 108, 111. In 21 to 55 percent the precipitating factor is contact lenses and infectious keratitis in these cases is more prone to be originating from Gram negative bacteria, often represented by Pseudomonas species 108-111, 114. Corneal infections caused by such strains are recognized for high virulence in addition to complex pathogenesis, which are enabled not only through several structural features but also by excretion of different factors, like toxins and multiple enzymes 115-117. Consequently, Gram negative bacteria are comparatively common as the causative agent in the most complicated cases of infectious keratitis, resulting in corneal perforations, endoftalmitis, or loss of the affected eye 118-

121. Lens wear has been identified as one of the primary risk factors associated with the very severe, still relatively uncommon, form of keratitis caused by the ubiquitous and predominately water-living protozoa, Acanthamoeba 122-126. A corneal infection due to this pathogen is routinely treated with a combination of drugs, administered topically at frequent intervals often for a period of months 125, 127. Moreover the therapy is often linked to undesirable side-effects, such as burning sensation in the eye or epithelial damage.


Figure 6: SEM picture depicting an Acanthamoeba trophozoite. (Reprinted from Marciano-Cabral F, Cabral G. Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev. 2003 Apr;16(2):273-307.)

Ulcerative keratitis on the basis of fungi is primarily encountered in tropical areas 128-130 and is analogously to Acanthamoeba keratitis often contact-lens associated. This type of infection can also be refractory with potentially detrimental outcome for future vision of the infected eye 131-133. Both fungal and Acanthamoeba keratitis can be highly complex to manage as the antibiotic therapy arsenal is limited and the inherent properties regarding these groups of microbes make them highly resistant.

The microbes isolated most frequently on sampling from corneal ulcers demonstrate the presence of bacteria such as Staphylococcus epidermidis, Staphylococcus aureus, Streptococci, and Pseudomonas aeruginosa, however most epidemiological studies present a relatively large proportion of negative culture results.


Antibiotic Resistance

The consequences of antimicrobial resistant microorganisms are undoubtedly of enormous proportion for clinical health care as well as socioeconomics, and the estimated cost concerning this issue, solely in health care settings situated in the United States, lie in the region of billions of dollars 134. Patients infected with antibiotic resistant bacteria have longer hospitalization duration, higher costs, and increased mortality relative to patients with susceptibility to therapeutic option, even though the virulence of the pathogenic strains does not necessarily differ 135, 136. Examples of some resistant microbes of greater consequence are methillicin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), and Gram negative bacteria with ESBL (Extended Spectrum β-Lactamase) activity. ESBLs are plasmid-mediated enzymes with the capability of degrading Beta-lactam antibiotics, hence giving the bacteria protection from the antimicrobial drug. Klebsiella species, like K. pneumoniae, and Escherischia coli are frequently isolated strains with ESBL-producing properties but other bacteria like Pseudomonas with ESBL have been identified 137, 138. One example of the spread of antibiotic resistance is MRSA, which today accounts for more than a quarter of staphylococcus bacteremia in parts of Europe 139.


Figure 7: Spread of MRSA in Europe 2009. Figure 8: Fluoroquinolone-resistant Pseudomonas aeruginosa in 2009 (Images in Figures 7 and 8 courtesy of the European for Disease and Prevention Control. Antimicrobial resistance surveillance in Europe 2009. Annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net))


Perhaps the most alarming examples of pathogens are however those isolates not responsive to several or any antibiotic agents, referred to as multi- respectively Pan-drug-resistant bacteria; features recognized predominately among Gram negative strains 140-143. Regarding microbial sampling isolates originating from ulcerative keratitis there are trends towards increasing ratios of antimicrobial resistance bacteria, although variations exist in resistance patterns over time 144-146. Susceptibility to Chloramphenicol particularly in Gram negative species have been described as low in several reports 147-150. Furthermore, fluoroquinolone resistant bacteria account for a considerable proportion of microbial culture results 144, 145, 151, 152, also predominantly among in Gram negatives. In some cohorts ciprofloxacin-resistant Pseudomonas isolates reach levels of 9 %. The novel (fourth) generation of this group of drugs (gatifloxacin and moxifloxacin) is more potent in elimination of Gram positive microbes but the efficacy is equal in eliminating Gram negative species compared to earlier drugs from the same category 152, 153. The recently introduced fluoroquinolone, besifloxacin, has however shown a higher capacity compared to older generations regarding bactericidal activity 154 and in experimental pseudomonas keratitis 155, making it a possible new therapeutic option upon encountering antimicrobial resistance. Examples of isolates originating from ocular infections have been documented with antimicrobial resistance regarding Gram negative ESBL- mediated resistance, towards several third-generation cephalosporin antibiotics 156 and resistance to multiple antibiotics, including fourth generation fluoroquinolones 157-159. In view of the above, the time consumption, complexity, and expenses for development of new antibiotics collectively make pathogen antimicrobial resistance an indisputable clinical concern, which could impede future infection therapy considerably.


Structure of the Cornea Figure 9: A sagittal section of the eye. The cornea is localized far right on the illustration. The cornea is shaped like a dome and is one of the few transparent tissues of the human body. In the periphery the stroma is thicker than centrally, however the thinnest portion of the cornea is located slightly infero-temporally 160, 161. Under normal conditions the thickness of the central cornea ranges from 520 to 550 microns 162, 163. A five to seven-layered structure of non-keratinized stratified squamous cells, with a thickness of roughly 50 microns, forms the epithelium 164, 165. Apart from preserving the tear film which keeps the eye moist, the epithelial surface is also of importance in protecting the ocular surface 166. The underlying layer is called Bowman’s membrane, an acellular structure, without capacity for regeneration 167. Constituting approximately 90 % of the cornea, the stroma and is localized between Descemet’s membrane, towards the endothelial side (posteriorly/basally) and Bowman’s membrane, towards the epithelial side (anteriorly/apically), with a thickness ranging from 450 to 500 µm 168. The stroma is composed of regular collagen fibrils (mainly type I collagen) 169 with similar sizes, arranged into lamellar structures forming the tissue; an arrangement which enables corneal transparency 170.


Figure 10: Ultrastructural view of the corneal lamellas composed of collagen fibrils. (Reprinted from Komai Y, Ushiki T. The three-dimensional organization of collagen fibrils in the human cornea and sclera. Invest Ophthalmol Vis Sci. 1991;32:2244-2258. Copyright holder ARVO, The Association for Research in Vision and Ophthalmology) Spread in between the fibrils keratocytes are localized which produce and maintain the extracellular matrix, primarily composed of glucose-amino-glycans (GAGs) 171. These cells are also an important source of matrix metalloproteinases (MMPs) that are involved in the stromal environmental equilibrium, remodelling of the stroma, and healing in corneal ulceration 172. In vivo measurements of the cellular density in the stromal tissue have confirmed a higher number of cells, keratocytes, in the anterior than in the posterior part 165, 173, 174. Descemet’s membrane is a basement membrane for the endothelial cells, forming the innermost layer of the cornea, facing the anterior chamber. These columnar, hexagonal cells, arranged in a single-cell layer are highly active metabolically and vital for the maintenance of the stromal hydration balance 175. Both the numbers of keratocytes and endothelial cells are reduced continuously during life 176-178 but an abnormal reduction of the endothelial cell density leads to an insufficient endothelium


function. Hence, a corneal edema and subsequently a loss of corneal transparency follow. This can occur from hereditary corneal dystrophy, trauma, or iatrogenic cell-loss as a result from intraocular surgery 175. A complex network of nerve cells mediates the corneal sensitivity, but is also believed to be of imperative meaning for the different corneal cellular functions regarding regulation of homeostasis and responses to different trauma. An arrangement of nerves, nerve bundles, and plexi throughout the different strata of the cornea constitutes a system which makes the cornea the most innervated surface tissue of the human body, indicating the importance of this aspect 179.

Corneal Immunology

In 1948 it was first proposed that the eye is one of the human organs classified as an immune privileged site 180, along with the brain, testis, and pregnant uterus (maternal/fetal interface). This signifies that a foreign tissue graft, which in other tissues is rejected by an immune reaction, placed in such location, is not followed by an inflammatory response and subsequent graft rejection. The privilege comprises inhibition of the immune system in the intraocular environment and downregulatory functions regarding inflammation as well as development of tolerance to antigens originating from the ocular tissues. The relatively high success rate of acceptance in corneal transplantation is believed to be partly a consequence of the host eye being a privileged site in addition to the donor transplant having immune privilege tissue properties 181. The ocular immunosuppression is thought to minimize the risk for collateral tissue damage due to excessive inflammatory reaction in the resistance against pathogens, hence, the absence of a number of immune cells customarily involved in the struggle against microorganisms elsewhere. The immune response of the eye is specific, mediated in part by cytotoxic T cells, with the capability to surpass the barrier of tight junctions between the blood stream and the intraocular environment as well as non-complement activating antibodies 182. Transparency of the cornea is vital for the refractive functions of the eye and the corneal response to pathogens is directed at minimizing the risk for scar-development and neovascularization, which could influence the visual acuity negatively. Antigen-presenting cells (APCs), such as epithelial Langerhans cells (LCs) and stromal dendritic cells (DCs), are important for the functions of the immune response, furthermore the latter group of cells consists of a diverse collection of immature and mature DCs, indicating the potential for dynamic actions against intruders. The layers of the cornea are also populated by tissue macrophages at different levels providing further defence against microbes 183, 184. Beside their


phagocytic activity, these also secrete cytokines, important for the recruitment of other immune cells in cases of a bacterial infection 185, 186. Host response in bacterial keratitis involves an upregulation of multiple inflammatory cytokines (like IL-1, IL-6, and TNF-αα), the recruitment of phagocytic immune cells, and elimination of microorganisms through different mechanisms 187. The outcome along with complications in corneal infection is influenced by multiple factors, such as the specific virulence and pathogenicity of the causative microorganism. Also, the complex interaction between the different corneal immune cells and different cellular responses to intruders are of importance 188-191.

CXL in Keratitis

Experimental Investigations Collagen degrading enzymes play a central role in corneal melting, a feature which is associated with both infectious and sterile keratitis. The ability of collagenases to degrade the collagen of porcine corneas is impaired by CXL and the procedure elevates the resistance against enzymatic digestion substantially 21. Similar results have been reported regarding chemical crosslinking by genipin and its inhibitory effect on the action of bacterial collagenase 192. The antimicrobial efficacy of riboflavin excitation has been well established in the research regarding the pathogen reduction technology for transfusions, where a number of microorganisms have been efficiently eradicated through the process (see separate Introduction). There are however some differences between the two photooxidative methods realizing the activation of vitamin B2 using ultraviolet light, of which the most imperative may be the variations of the wavelengths exploited. In experimental ophthalmological research the interaction mediated by the particular light spectrum employed in CXL has been investigated through illumination of agar plates with different bacteria, concluding that the method has an antibacterial capacity 193, 194. Furthermore, the joint action of vitamin B2 and ultraviolet light can potentiate the elimination of different fungi subjected to Amphotericin B 195, supporting the hypothesis that the oxidative stress induced by the process has an antimicrobial effect.

Clinical Results The first account of riboflavin-UVA treatment of corneal infections was published in the year 2000, where it was reported that four clinical cases had received treatment against corneal ulcers with melting of different origins. Of these three healed after the therapy 196. Several case reports have since described curing of non-healing ulcers 197, of corneal melting, and


recalcitrant corneal infections after conducting the CXL procedure 198-203. Only a limited number of these publications have reported more than single cases and presently there is a discernible lack of prospective clinical studies. Applying the protocol for keratoconus, 6 out of 14 non-healing ulcers were cured following the therapy 197. Another article described five cases of recalcitrant microbial keratitis, including corneal melting which all were treated successfully through the implementation of the CXL procedure 201. Moreover, it enabled management of 3 eyes suffering from refractory Acanthamoeba keratitis in another report 202.


Aims

• To evaluate whether the photoactivation of riboflavin using the parameters employed in Collagen Crosslinking can eliminate different bacteria commonly isolated as pathogens in microbial keratitis.

• To compare differences in the microorganism response subjected to the photochemical process.

• To ascertain if riboflavin is required for the antimicrobial effect or if ultraviolet light solitarily is sufficient.

• To infer if the concentration of riboflavin is of importance for antibacterial efficacy and if so, to further attempt determining at which concentration the eradication peaks.

• To study the growth of the protozoa Acanthamoeba castellanii exposed to riboflavin/UVA and to assess if any growth inhibition can be attributed to UVA illumination alone or if an interaction effect between the factors take place.

• To describe the clinical effect of Collagen Crosslinking in severe cases of infectious keratitis.

• To investigate the clinical effect of the photochemical interaction, utilized in Collagen Crosslinking, as a primary therapy for bacterial keratitis.


Patients The patients involved in paper I were retrospectively recruited from the Department of Ophthalmology in Örebro. They constituted all patients with a clinical diagnosis of severe infectious keratitis treated by CXL, between February and August 2008, where adequate photographic documentation during the episode of infection and at follow-up visits existed. In paper III prospective recruitment of patients was conducted from the Departments of Ophthalmology in Örebro and Jönköping. Consecutive recruitment was performed of all patients eligible according to the study protocol, notified to the investigators. Study subjects with an episode of suspected bacterial keratitis, without prior antibiotic therapy were included and received treatment with CXL as primary and solitary therapy for the infection.


Materials and Methods Case Series (Paper I) A retrospective review was made of patient journals of subjects having received therapy for severe infectious keratitis by CXL between February and August 2008. Inclusion was made of all patients where diagnosis and follow-up could be supported by photographic documentation during the episode of infection and after healing of the ulcer. The duration of follow-up after the adjuvant photooxidative therapy ranged between 1 to 6 months after initial presentation. The purpose was to describe the clinical response observed after the CXL procedure, which enabled management of the infected ulcers. Experimental Eradication of Bacteria in Fluid Solutions (Paper II) Bacterial reference isolates of Staphylococcus epidermidis, Staphylococcus aureus, and Pseudomonas aeruginosa were cultured on blood/hematin–agar plates. The microbes were subsequently dispersed in PBS fluid, to a concentration of approximately 108/ml. Determination of microbial number was conducted through counting in a Burker chamber. Stock solutions of each bacterial strain were prepared through dispersion in PBS, to end concentrations before illumination of concentrations of 2-5×105/ml. In order to avoid disturbance of light absorption or scattering, uncolored fluids were selected for the irradiation. The experiments were performed at different riboflavin molarities (concentrations), ranging from 0 to 400 μM, with the purpose of isolating at which point the bacterial elimination was most extensive. Methodologies described by Martins et al.193 and Schrier et al.194 could not address this issue since exposure in both experimental settings were done on agar plates. A thin fluid layer was desired in the vessels, however maintenance of layer integrity during the entire experiment was vital, why 100 μl was selected. The entire surface was exposed to UV light, wavelength 365 nm for 30 minutes (dose 5.4 J/cm2), with following determination of CFU (Colony Forming Units)/ml. Thereafter the UV irradiation was repeated, resulting in a doubling of the UV dose, and CFU/ml assessment was iterated. Every fluid preparation subjected to ultraviolet light had an unexposed (negative) control solution, originating from the same source suspension. All measurements were executed in triplicates. Statistical analysis involved two-way ANOVA as well as unpaired t-tests at each measured molarity, comparing the differences between UV-exposed/unexposed measurements.


Pilot study (Paper III) A clinical pilot study was conducted to investigate CXL as the primary and solitary therapy for bacterial keratitis. The study protocol was established in association with the Clinical Research Support (CRS) Centre, Örebro University Hospital. Authorization was granted from the Regional Ethical Committee as well as from the Medical Products Agency for Clinical Trials. In order to isolate the effect of the CXL procedure no antibiotics was given during the trial, unless tendencies for infectious progression were detected, and only patients without prior antibiotic treatment for the current episode of infection were eligible for inclusion. Therapy with CXL was conducted in patients fulfilling all the inclusion as well as none of the exclusion criteria and before receiving results from microbial culturing, since the subjects could not be left without treatment during the isolation of the infectious agent. After the procedure patients were closely monitored, initially through daily examinations and after signs of improvement at a reduced frequency. Patients were hospitalized if needed. Slit-lamp photography was carried out at each follow-up visit, or if hospitalized once daily. The patients were given antibiotics if any signs of deterioration occurred. Regular examinations were continued until the ulcer had healed and all symptoms had regressed. Growth Inhibition of Acanthamoeba (Paper IV) Acanthamoeba castellanii was cultured and prepared into fluid solutions in a similar setup as paper II. The exposure of UVA was carried out using a analogous procedure, however since the growth of the protozoa is considerably slower than bacteria examined in the previous experiments, determination of the microbe number was made 4 to 7 days after exposure. Instead of selecting one point of time for the determination of microbial proliferation a growth curve was plotted, including the negative non-exposed solutions. The concentration of riboflavin chosen for the experiments was 0.01% as it was an efficacious concentration in bacterial elimination tests and preparatory experiments for Acanthamoeba indicated no major variations when riboflavin concentration was altered. In these experiments a repeated-measurement ANOVA for unpaired samples was used as well as post hoc analysis regarding pairwise comparisons. Surgical Technique (Papers I and III) The surgical techniques involving patients in papers I and III were similar. Under topical anesthesia, loose epithelium surrounding the corneal ulcer was removed, using a sterile swab. Riboflavin administration was carried out for 20 minutes (in paper I, up to 30 minutes) at an interval of every 2-3 minutes during the entire procedure. Illumination was done using the settings for


keratoconus (3.0 mW/cm2 for 30 minutes, dose 5.4 J/cm2). In one patient (paper I) with an advanced non-responsive bilateral keratitis, with multiple risk factors for microbial keratitis, the photochemical procedure was followed by a human amniotic membrane transplant in both eyes. The riboflavin used differed between paper I and III, in the former isotonic riboflavin dextran solution (Medio-Cross riboflavin/dextran, 0.1%) was used and in the latter 0.1% isotonic riboflavin (Ricrolin®) was administered. UV illumination devices (Papers I through IV) Papers I through III utilized the UV-X™ (Peschke Meditrade GmbH, Switzerland), 365 nm, as light source for irradiation. Calibration of the device was made before implementing the procedure involving patients and prior to experimental illumination in paper II. Distance from the surface was, as recommended by manufacturer, measured to 5 cm from the cornea/solutions. In the in vitro experiments involving Acanthamoeba castellanii the Opto Xlink-Corneal Crosslinking Systems nr 141 (365 nm) was used, operating at a distance of 4.5 cm from protozoal suspension. The effect was 6.366 mW/cm2 and the exposure time 14 minutes and 20 seconds, resulting in a UV dose of 5.475 J/cm2. Microbial Sampling (Paper III) The isolation of possible causative microorganisms in paper III included conjunctival smears as well as corneal sampling. The cultures and scrapings originating from the cornea were incubated on blood agar, Sabouraud agar, and GC agar. Sterile scalpel blades were also placed in broth as well as Page’s solution to enable detection of Acanthamoeba.


Results and Discussion Paper I Seven eyes of six patients were included in the case series and all eyes healed after the given therapy. Improvement of symptoms, including reduction of associated inflammation and pain, occurred within 24 hours postoperatively. Regression of both cases with visible hypopyon took place 2 days after the treatment. Associated corneal melting did not progress in the treated subjects after therapy. The ulcer size ranged between 1 mm in diameter and engagement of the entire cornea. Bacteria on microbial cultures were detected in four of the cases, comprising both Gram-positive and negative strains. At last visit the epithelium was intact and no inflammation was seen in any of the treated eyes. None of the patients was subjected to an emergency keratoplasty. The duration of the epithelial healing was variable and took place within a time period of one week up to several months. The mean visual acuity had at latest follow-up examination improved from 0.06 (range, light perception to 0.2) at diagnosis of infection to 0.20 (range, hand motion to 0.6). Riboflavin photosensitization in therapy of infectious ulcers was first described by Schnitzler et al. who published an article of four patients with corneal melting from different etiologies treated successfully in three cases, by employing the treatment later referred to as CXL 196. In support of this Iseli et al. reported five cases with corneal melting associated with microbial keratitis that all healed after treatment with the same procedure, unresponsive to the preceding antibiotics given 201. Other groups have accounted for case reports of keratitis responding to the therapy with cure as an adjuvant method to antibiotics in refractory cases. The majority of the cases were of advanced nature, with large corneal ulcers, intense associated inflammation, and risk factors for complicated courses of infections. After conducting the CXL procedure all eyes healed without complications. Consistent findings postoperatively were a marked symptom improvement, reduction of inflammatory level, and initiation of epithelial healing shortly after the procedure. In view of the severity of cases, with non-responsiveness to the given therapy in three eyes (case 1 and 6), multiple risk factors in most cases, and one patient healing without receiving antibiotics after suture removal from the suture-related infectious ulcer the article supported previous reports of microbial keratitis successfully managed by CXL.

An unanticipated feature after therapy was the quick improvement in symptoms like pain, light sensitivity, and associated inflammation, with rapid abolishment of hypopyon postoperatively, in both of the eyes where it


was observed at presentation. This could be explained by different, perhaps synergistic mechanisms. The riboflavin photosensitization by UV light is an established method to generate reactive oxygen species and eliminate microorganisms, as is exploited in the pathogen reduction technology for transfusions. Eradication of etiologic microbes could therefore be the reason for the alleviation of symptoms and inflammation. Inhibition of corneal sensation, due to a direct effect of riboflavin or oxidative processes could also be a factor explaining the observed symptom reduction, however not regarding the inflammatory response to the method. Modulations of the ocular inflammatory response, by affecting the activities of corneal immune cells such as Langerhans and dendritic cells, is another aspect which could be of importance for the abolition inflammation coupled with the infection. The riboflavin photoactivation in transfusion medicine has, besides the elimination of pathogens, also a documented inhibitory effect on white blood cells, and as a consequence could be utilized to prevent transfusion-associated GVHD. One could hence hypothesize that a similar inhibitory effect on the corneal immune system takes place when applying the CXL treatment.

There are other factors which also could be of value in resisting a corneal infection and corneal melting. Spoerl et al. have described a considerable increased resistance against collagenases in corneal tissue subjected to the UVA-riboflavin treatment21. Since enzymatic degradation is involved in, and considered as a central aspect of, corneal melting this property is another relevant point which could be protective in both infective and inflammatory keratitis. The induction of apoptosis among keratocytes as well as the direct structural alterations of the corneal stroma, caused by the crosslinking therapy could also modify the environment for the causative pathogens, consequently obstructing optimal conditions for microbial proliferation and tissue infiltration.

In conclusion, the collective observations of conducting CXL in these cases of infectious keratitis suggest that photoactivation of riboflavin through implementation of the procedure could be considered in severe and recalcitrant cases of corneal infection. We construe this from the pronounced response to therapy observed regarding subjective symptoms, associated inflammation, and corneal melting among all the treated patients. Taking into account that the procedure is regarded as safe with low frequencies for postoperative complications, it seems reasonable that an introduction for its use is possible, as an adjuvant therapy in corneal infection and melting. A marked stromal thinning due to melting will naturally compromise the safety regarding the endothelium; however in a situation with progressing corneal degradation the risk for endothelial cell


damage must be balanced against other alternative options, such as an emergency keratoplasty. Paper II Staphylococcus epidermidis UVA and riboflavin united at the dose utilized in clinical CXL (5.4 J/cm2) resulted in a tendency towards decreased numbers of Staphylococcus epidermidis, however this reduction could not be confirmed by statistical significance. Upon doubling the UV level a decrease up to 90 % was achieved and at all tested riboflavin molarities statistically significant reductions were confirmed. A bactericidal effect was not detected by ultraviolet light exposure alone at either of the tested UV levels. The interaction term between riboflavin and UVA was statistically significant (p=0.008). Staphylococcus aureus This strain showed a growth pattern similar to the previously investigated bacteria, however the drop in CFU count could already at the lower UV dose be verified statistically, with the exception of the reduction at 300 μM of riboflavin. At the elevated dosage of UVA up to 71 % of the microorganisms were abolished, with a tendency towards a more complete eradication at higher riboflavin levels. The UVA riboflavin interaction term was significant (p=0.038). As with Staphylococcus epidermidis, the ultraviolet light solitarily did not result in a significant bacterial elimination. Pseudomonas aeruginosa The bactericidal efficacy regarding Pseudomonas displayed a consistent tendency towards elimination of the bacterial strain at all tested riboflavin concentrations. As the variations in the CFU counts were greater in this bacterium than the two preceding strains, the effect could only be detected statistically at 300 μM, using the lower UV exposure settings. The outcome of the augmented dose, however, accomplished an almost absolute sterilization of the fluid solution at all tested molarities, ranging from 98 to 100 %, with a statistically significant interaction term (p=0.034), suggestive of the result being a consequence of the combination of UVA and riboflavin. No indications towards an antimicrobial action of the ultraviolet light single-handedly were observed at the tested irradiation levels. In the in vitro pathogen eradication experiments of common etiological bacterial strains isolated in microbial keratitis we could support the earlier observations of Martins et al. 193 and Schrier et al. 194 that the UVA


photoactivation of riboflavin has a bactericidal effect. These previous laboratory investigations could, due to the experimental setup, not determine at which riboflavin concentration the pathogen elimination was most efficacious. Through the experimental design using bacteria in fluid solutions it could be established that only a surprisingly low quantity of the vitamin was needed for microbial inactivation. This indicates that under clinical conditions it is plausible that the stromal concentration of riboflavin is sufficient for achieving an antimicrobial effect. Responses to the evaluated photooxidative insult varied considerably in the different strains of bacteria, principally when comparing Pseudomonas aeruginosa to the two Staphylococcus species. The differential outcome could perhaps be attributed to differences in structure or metabolism. Regarding the anaerobic bacteria an almost complete eradication after illumination was displayed at the higher UV dose tested, signifying a higher sensitivity to the UVA-riboflavin interaction. Since Pseudomonas aeruginosa, aside from its recognized virulence, is prone to develop resistance to antibiotics the oxidative stress induced by vitamin B2 photosensitization could be especially appealing in addressing these bacteria during corneal infections. In all tested bacteria the concentrations evaluated pointed to peak eradication at low levels of the vitamin, somewhere in the region of 0.01 % or below. Elevating the riboflavin concentration did not increase depletion of microorganisms but instead seemingly reduced this outcome. We hypothesize that excessive riboflavin can shield bacteria in the depths of the solution thus reducing the effect on microbes, an indication that in exploiting the photosensitization procedure clinically one should be aware of not allowing a thick layer of riboflavin cover the cornea during illumination.

Nevertheless, we could demonstrate that riboflavin is vital for the antimicrobial result in all the tested strains and that ultraviolet light single-handedly did not eliminate the bacteria. It could be concluded that the interaction of ultraviolet light and riboflavin was the causative factor in these experiments and neither isolated riboflavin nor UVA at the doses elucidated could explain the impact on bacterial reduction. It is likely that the oxidative stress generated by UVA-riboflavin causes the depletion of pathogens observed, which is also the mechanism exploited in photodynamic therapy (PDT) for infections. The generation of ROS and hydrogen peroxide are consequences of riboflavin excitation and credible contributors to the elimination of bacteria, both under the laboratory settings and under clinical conditions. Although the practise of vitamin B2 photoactivation in transfusion medicine, employed by the Mirasol® PRT system, has a wider spectrum of ultraviolet light, including lower wavelengths than in CXL, the mechanisms of


oxidative injury are likely to be the same. The intercalation of the planar part of the riboflavin molecule into the genetic material of microorganisms is with high probability identical and the non-specific oxidative damages should be similar, however the efficacy of the shorter ultraviolet spectrum may well be superior to the single wavelength of 365 nm exploited in CXL. The capacity of the Mirasol® device in reduction of pathogens has been explored in detail with the potency to eliminate viruses, bacteria, and parasites effectively. It is on the other hand probable that the human tissue response, in this case the cornea, would be altered with differences regarding safety aspects and degree of corneal apoptosis, if the same ultraviolet spectrum would be applied as an infectious treatment in keratitis.

Given differences in the bacterial responses to the oxidative insult evaluated the specific effect should be ascertained in more bacteria to establish which pathogens are more and less susceptible to the process. It is however important to emphasize that even though the number of bacteria were few, all the analysed strains reacted to the procedure, and these pathogens together consistently represent a substantial proportion of infectious keratitis in clinical epidemiological studies.

The investigational model developed for these experiments involved a fluid layer with a theoretical thickness of over 1.75 mm at the start of illumination, assuming a planar surface of the solution. The evaporation of water during the irradiation did not interfere with fluid-layer integrity during the whole experiment; yet, surface tension inhibited the use of substantially smaller volumes. Due to the limited penetration of the particular wavelength, it is probable that the entire liquid strata were not fully exposed to the UVA, even when considering that tension forces between the fluid and inner wall of the well, most likely resulted in a slightly thinner level centrally, compared to the theoretical planar distribution. In the clinical situation apoptosis is mostly seen in the anterior 200-300 microns, implying that the effect is reduced in the deeper strata of the cornea. Analogously, it can be assumed that in the investigational setting the lower levels of the fluid layer was not exposed to the same UV dose as the more superficial parts of the bacterial solution, resulting in an impaired exposure at the base of the experimental well. It is thus reasonable to estimate that the antibacterial efficacy in fact may be underestimated in our model, and could be one explanation why the clinical UV dose of 5.4 J/cm2 resulted in only a minor reduction of microbes. These in vitro eradication experiments should therefore be supported by, and evaluated further through, tissue and in vivo experimental models for keratitis to establish the bactericidal efficacy clinically. Perhaps it could be argued that more appealing systems for isolating the antimicrobial efficiency of the process in


question is a system of micro-wells or fluid films, where the thickness of fluid solutions could reach below 300 microns. It is noteworthy that the investigation of bactericidal capacity comprised pure riboflavin, and not riboflavin-5’-phosphate, also known as flavin mononucleotide (FMN), which is the substance used in clinical crosslinking, according to the standard protocol. The biological effects of these compounds are assumed to be similar although not necessarily identical. Since the absorption spectra of the molecules are comparable and the planar molecular part one and the same, it seems plausible that divergences in the clinical outcome should not be of greater magnitude regarding bactericidal efficacy. As differences in water solubility, molecule degradation time, and tissue diffusion could influence the microbial response the effects of each compound should in the future be elucidated separately and compared.

Paper III Of the 16 subjects included in the trial all exhibited a clinical response towards the procedure, with a reduction in inflammation and initiation of epithelial healing. The primary study end point was thus realized in all patients. Epithelial healing of the cornea after the CXL therapy occurred after one to 14 days postoperatively (mean 7.1) and visual acuity increased in seven patients and decreased in one. In one eye, with a corneal edema before the start of the infection due to rubeotic glaucoma, an amniotic membrane transplant was conducted to facilitate epithelialisation. Two patients required antibiotic therapy during the course of the infectious episode, one due to recurrence of an epithelial defect after initial healing and the other because of development of a new ulcer during the course of epithelial recovery. The latter of these had a bandage contact lens (BCL) at the time of the secondary ulcer. No side effects or therapy complications were seen during the course of the study. The microbial culture results were positive for bacteria in thirteen of the subjects, comprising Gram-stain positive and negative strains. To our knowledge the protocol assembled for the pilot study evaluating the photochemical interaction utilized in CXL as therapy for bacterial keratitis is the first published prospective study worldwide on the topic. The trial investigated the procedure as the study intervention, under careful and frequent assessment of clinical status during healing, in addition to the possibility of incorporating antibiotics at any stage of the study if deterioration occurred. Our particular study model was selected in order to isolate the clinical effect of the method and drawing conclusions regarding its ability to induce healing of microbial keratitis, with a limited number of


study subjects. Although a randomized study protocol would have elevated the strength of the trial it would have undoubtedly required a significantly larger study population. The purpose was not to assert CXL superiority versus antibiotics but to demonstrate and evaluate the efficacy of the procedure in treating corneal infections. Considering the rate at which antibiotic resistance is spread globally, the comparative lack of antibiotic development, and absence of antimicrobial drugs regarding some microorganisms, introduction of a new approach in keratitis therapy utilizing different mechanisms than antibiotics may be of great value in management of corneal and perhaps other types of infections.

Three patients needed additional therapy during the course of the infection. One chronic ulcer, due to a corneal edema, required an amniotic membrane transplant and two patients received topical antibiotics. In one of the subjects where antibiotics was necessitated the infiltrate was particularly deep, reaching the level of Descemet’s membrane. Even though hypopyon was cleared in addition to healing of the epithelium shortly after CXL, the inflammation did not completely regress and 14 days after presentation a slight epithelium defect recurred. We interpreted this as an indication of limited effect in treatment of infections engaging the deeper strata of the corneal stroma. In these cases it is still not certain that UVA-riboflavin photochemical therapy is inappropriate since reduction of the microbial number in more superficial parts of the cornea may be of value, as well as utilizing the other mechanisms that appear to be involved in healing of a corneal infection treated with the method.

In addition to the absence of a control group, the study limitations involve a limited number of patients, predominately small corneal ulcers, and a relative uncertainty regarding the diagnosis of bacterial keratitis. A small infectious infiltrate would naturally be easier to handle with lower risk for a complicated course of the disease. More advanced cases of keratitis could have affected the outcome of this trial possibly resulting in a larger number of treated eyes receiving antibiotic treatment. The cases in the study were however not selectively included, but consecutively with an active intent of limiting exclusion criteria to a minimum. Regarding the etiologic microorganism in each case, it is feasible that colonizing bacteria in some cases could be the isolates considered as the pathogens. Perhaps even some of the cases diagnosed as a bacterial keratitis were in fact non-bacterial ulcers. Even so, it is unlikely that a considerable proportion of the patients included were of this nature and all the ulcers responded to the therapy, pointing to efficacy regardless of causality. A proportion of culture-negative ulcers are consistently reported in different publications. In one trial subject the corneal culture results displayed growth of bacterial strains resistant to


fluoroquinolones and aminoglycoside antibiotics. At the time of microbial sampling results the ulcer had already healed, demonstrating the risk for infectious progression of the ulcer during the elapsed time period if customarily prescribed drug treatment had been administered. Antibiotic resistance does probably not indicate an inherent protection against photooxidative stress.

In contrast to antibiotics the assumed multiple action of the CXL procedure makes it more appealing as a complementary therapy to antibiotics in more severe cases of infection and if administered antibiotics are futile. Considering the fact that corneal melting associated with microbial keratitis can be rapid, one could argue about at which time point the illumination treatment should be done, if implemented in management of infections. The chances of avoiding an emergency intervention are probably lower if the clinical deterioration is allowed to continue. As the therapy has not yet been thoroughly investigated for this application, it appears reasonable that it should be practised with caution and in cases where prognosis with the regular therapy is estimated to be poor or when treatment refractory infections are confronted.

This study was consistent with the previous clinical article (Paper I) in the rapid response and symptom improvement shortly after the procedure. The trial outcome demonstrates, and further supports our earlier findings, that the photosensitization of riboflavin is an approach that can elicit healing of infectious keratitis, hence having an effect in treatment of this condition. Paper IV The growth of Acanthamoeba castellanii subjected to ultraviolet light was impaired matched to the increase in microorganism number found in unexposed solutions over the measured time period (days 4 to 7), with a decrease in pathogen count of 20 percent as a consequence of illumination with UVA dose of 5.475 J/cm2. The addition of riboflavin in solutions did not amplify the antiprotozoal efficacy. In the evaluation phase after UV exposure at the higher dosage a more manifest inhibition of elevation in pathogen count was observed. In contrast to the findings from the previous series of experiments (Paper II) the combined UVA/riboflavin displayed no indications of an interaction effect but the antimicrobial action regarding protozoa seemed to be mediated solely by ultraviolet light. The repeated-measurement ANOVA confirmed the association of the UVA effect (p=0.001). Pairwise post hoc evaluations were statistically significant for the abolition of protozoa, concerning the solutions subjected to the higher UV dose (p=0.008) but not to the lower (P=0.090), consistent with a dose-


response relationship. Riboflavin showed no tendency signifying an antiprozoal action by itself or grouped with UVA, which was supported by the ANOVA analysis (p=0.992 and p=0.977 respectively). Although Acanthamoeba is known to be resistant to UV light, it has been established that both trophozoites and cysts can be eliminated by ultraviolet light exposure. Since reported clinical cases of recalcitrant keratitis caused by the microorganism have healed after treatment with UVA-riboflavin photosensitization 202, 203 we intended to evaluate the responsiveness of Acanthamoeba castellanii to the phenomenon, by investigating its potential to induce growth inhibition of the parasite. Assessment of pathogens on the final day of the experiment displayed a reduction in the number of protozoa with similar efficacy when subjected to ultraviolet light and UVA-riboflavin. No indications were seen of an interaction between the factors supporting the view that riboflavin augments the effect of the irradiation in vitro. It could therefore be possible that ultraviolet light itself, in the reported cases, triggered healing of infections. Under clinical conditions however, when therapy with antiprotozoal drugs customarily has preceded the photochemical treatment, the inhibitory or elimination capacity may be different. Drugs, like chlorhexidine and polyhexamethylene biguanide (PHMB), damaging the cellular membrane or cell wall of the protozoa could for example facilitate uptake of the vitamin increasing its susceptibility to the oxidative stress generated by the procedure. A synergistic antimicrobial effect has for example been observed regarding UVA-riboflavin photoactivation in combination with Amphotericin B on fungi 195. Several other explanations are possible as the CXL procedure used clinically initiates structural and biomechanical alterations in the corneal stroma, which could influence both the growth of the parasite and the host response.

Even though the solitary UVA illumination of Acanthamoeba castellanii was equally or perhaps slightly more efficient regarding the antiprotozoal effect than ultraviolet light and riboflavin coupled, it is unlikely that it could be realized as a clinical therapy. Vitamin B2 absorbs the UV light passing through the stroma, thus reducing the dose reaching the endothelial level, furthermore protecting the inner structures of the eye, such as the lens and the retina. Absence of it would therefore compromise the safety of the procedure as a whole.

Since different articles investigating Acanthamoeba utilize diverse durations to establish the outcome or elimination of the pathogen we elected in our experimental model to measure the microorganism number over time, with a repetition of measurements. Count of protozoa was accordingly conducted on several consecutive days and plot of a growth curve over time


was done, instead of assessing microorganism number at one given point, with the purpose of increasing the reliability of the experimental procedure.

In conclusion, our investigations showed that UVA at 365 nm can inhibit growth of Acanthamoeba castellanii. The addition of riboflavin did not potentiate this effect, either in combination or single-handedly affected the growth of the protozoa significantly. The clinical response of treating Acanthamoeba keratitis 202, 203 could therefore be a consequence of the ultraviolet light itself, but due to the safety issues of CXL riboflavin is considered as a vital part of the clinical implementation of UVA-riboflavin photosensitization.


Conclusions • All bacterial isolates evaluated, represented by microorganisms

frequently found as causative pathogens in microbial keratitis, could be diminished significantly or eliminated by the UVA settings of 365 nm, which is exploited in CXL, if combined with riboflavin.

• The sensitivity to the photooxidative insult differed between different species and the most extensive elimination among the microbes investigated was observed in the tested Pseudomonas aeruginosa strain. A complete eradication of this strain could be achieved by the riboflavin –UVA interaction.

• In every bacterial strain the presence of the photosensitizer was vital to achieve the bactericidal effect and ultraviolet irradiation solitarily, at the wavelength of 365 nm, did not indicate a significant antimicrobial effect. Hence, the eradication of bacteria can be attributed to the joint action of ultraviolet light and riboflavin, through the oxidative stress generated by the procedure. This is in agreement with the photooxidative therapy mediated by the device for pathogen reduction in transfusion medicine.

• The required quantity of riboflavin was low and concentrations exceeding 0.01% seemed to achieve no further consequence regarding abolishment of bacteria.

• Growth inhibition of the protozoa Acanthamoeba castellanii by UVA illumination could be established but the interaction with riboflavin did not enhance the antiprotozoal effect, which signifies a diverging effect when comparing it with the bacterial elimination elucidated in previous experiments. This may be due to the structural differences between the different groups of microorganisms.

• UVA-riboflavin photochemical interaction utilized in Corneal Collagen Crosslinking (CXL) seems to be a method with properties sufficient to induce healing of severe keratitis and in some cases ulcers non-responsive to antibiotic treatment. The clinical effect also appears to involve an anti-inflammatory response to the treatment.

• To isolate and evaluate the clinical efficacy of UVA-riboflavin photosensitization, a pilot study was conducted with CXL as the


primary therapy for bacterial keratitis. 16 patients with the clinical diagnosis were included, of which 14 healed without antibiotics. No complications or treatment side effects were observed. Although the majority of ulcers in the trial were of limited size, the clinical effect regarding reduction of inflammation, symptom alleviation, and epithelial healing was clear, strongly indicating that the procedure has the capacity to induce healing of microbial keratitis. The purpose of the study however, was not to recommend CXL as a primary method but to establish that the method could be considered as an adjuvant treatment to antibiotics in selected cases, primarily where topical drugs may be insufficient. Riboflavin photoactivation in the management of microbial keratitis seems to be an efficacious option with a preserved high safety profile when utilizing CXL as a photodynamic therapy for corneal infections.


Populärvetenskaplig sammanfattning En infektion i hornhinnan (infektiös eller mikrobiell keratit) innebär att en mikroorganism, vanligen ett virus eller en bakterie, angriper hornhinnan (kornea). Majoriteten av odlingspositiva keratiter är bakteriella. Tillståndet medför risk för framtida synnedsättning och flera allvarliga komplikationer såsom uveit (inflammation i ögat), endoftalmit (infektion engagerande hela ögat) och till och med perforation, vilka kan utvecklas under förhållandevis kort tid. I sällsynta fall kan ögat på grund av infektionen till och med behöva opereras bort (så kallad evisceration). Den initiala terapin utgörs av intensiv droppning, i allmänhet med bredspektrumantibiotika. Efter erhållande av odlingssvar inklusive resistensbestämning kan man styra behandlingen till mera specifika typer av antimikrobiella läkemedel. Inneliggande vård är inte ovanligt, i synnerhet då mera avancerade fall, med större infekterade sår eller kraftigare inflammation, eftersom droppningen ofta måste ske intensivt dygnet runt den första tiden. Behandlingen sker ofta i några veckor men kan vid allvarligare typer av hornhinneinfektioner pågå i flera månader.

Antibiotikaresistenta bakteriestammar är ett allt större problem inom sjukvården och resistens finns redan idag även rapporterat mot i stort sett samtliga typer av idag tillgängliga antibiotika. Flera åtgärder har föreslagits för att begränsa spridning av sådana stammar till exempel restriktivitet av antibiotikaförskrivning, isolering av patienter med smitta och striktare hygienrutiner men trots så finns det tecken på att antalet resistenta mikroorganismer ökar. Detta skulle kunna innebära att infektioner ej kommer att kunna behandlas effektivt i framtiden med stora följdkonsekvenser för såväl vårdgivare som patienter. Önskvärt vore att finna nya sätt att angripa infektioner.

Riboflavin (Vitamin B2) som belyses med ultraviolett eller synligt ljus kan inaktivera flertalet virus, bakterier samt parasiter. Fenomenet är väl studerat och används inom transfusionsmedicin för reducering av patogener i blodprodukter. Den fotokemiska processen skadar mikroorganismerna dels ospecifikt, genom uppkomst av fria syreradikaler och dels genom att molekylen orsakar specifik skada på mikroorganismernas arvsmassa (DNA och RNA).

Samma princip används idag kliniskt vid Corneal Collagen Crosslinking (CXL). Detta är en behandling som utvecklats för att förstärka den försvagade hornhinnan vid tillstånd då denna är försvagad, så kallade ektasier, och på så vis vara ett alternativ till transplantation av hornhinnan. Behandlingen utgörs av en engångsbehandling med en kombination av riboflavin och belysning med ultraviolett ljus (UVA, våglängd på 365 nm).


En förstärkning av hornhinnan sker därefter som tros bero på uppkomst av nya korsbindningar mellan hornhinnans kollagenmolekyler. I experimentella studier har man visat att CXL hindrar kollagenasenzymers nedbrytning av hornhinnan och fotoaktivering av riboflavin med UVA har in vitro visats kunna eliminera olika bakterier. Kombinationen kan även potentiera avdödning av svamp. Man har redovisat fallbeskrivningar av korneala ulcerationer (sår) som läkt efter behandling med CXL och flera artiklar beskriver att man effektivt kunnat hantera svårbehandlade infektiösa keratiter med metoden.

Denna avhandling syftar till att utreda huruvida fotoaktivering av riboflavin kan tillämpas som ett nytt verktyg vid keratitbehandling samt att undersöka vilka faktorer som är av betydelse för detta.

I det första arbetet redovisades 7 allvarliga fall hos 6 patienter med infektiös keratit som behandlats med Collagen Crosslinking (CXL), som utfördes enligt standardprotokoll för korneal ektasi. Fotodokumentation av hornhinnesåret genomfördes och detta upprepades under uppföljningen. Med undantag av ett öga, erhöll samtliga infektioner behandlade antibiotika, utöver CXL-behandlingen. Alla patienter uppvisade en minskning av den korneala lesionen och reduktion av inflammatorisk aktivitet kort efter terapin. Nedsmältningsprocessen avstannade och en komplett läkning epitelytan skedde i samtliga fall.

För att utvärdera den antibakteriella effekten av behandlingen genomfördes en experimentell avdödning av tre vanligt förekommande mikroorganismer vid bakteriell keratit. Dessa odlades och inokulerades till en bestämd koncentration i ofärgat odlingsmedium. En mängd på 100 mikroliter applicerades i en brunn/kärl där riboflavin tillsattes i olika mängd (molaritet). Brunnens diameter valdes så att hela ytan kunde belysas homogent, vilket skedde med UVA i klinisk dos via diodlampa som används vid CXL-behandling. En liten mängd extraherades och inkuberades för odling, varefter UV-exponering med samma dos upprepades. Icke-belysta (negativa) kontroller fanns till samtliga lösningar. Bestämning av antal bakterier för de olika lösningarna skedde därefter. Man kunde fastslå att kombinationen av UVA och riboflavin hade en avdödande effekt på alla utvärderade bakterier. Endast en liten mängd vitamin krävdes för att uppnå effekten, som dock varierade mellan de olika stammarna. Belysning med enbart ultraviolett ljus ledde till en mycket begränsad, eller ingen eliminering av mikroorganismer.

I en klinisk pilotstudie genomfördes behandling av misstänkt bakteriella hornhinneinfektioner med den UVA-riboflavin som primär terapi. 16 patienter inkluderades vid Universitetssjukhuset i Örebro samt länssjukhuset Ryhov, Jönköping. Syftet var att under noggrann uppsikt utvärdera effekten


av CXL som terapi för bakteriella keratiter. Ingen antibiotika ordinerades innan behandling med CXL som skedde enligt standardiserat protokoll. Mikrobiell provtagning skedde enligt ett förfarande utarbetat tillsammans med mikrobiologiska kliniken, USÖ. Dagliga kontroller skedde tills tydliga tecken på utläkning observerats och därefter glesades dessa ut. Vid behov lades patienten in på avdelning för att möjliggöra mera frekvent undersökning. Minsta tecken på försämring innebar sedvanlig antibiotikaterapi. Kontrollerna av läkning pågick tills full epitelläkning skett och symptomfrihet uppnåtts. Av de inkluderade patienterna krävde två antibiotika för utläkning av infektionen. Odlingssvar från provtagning involverade både Gram-positiva och negativa bakterier.

I studiens fjärde artikel utreddes tillväxt av den svårbehandlade parasiten, Acanthamoeba castellanii, i vätskelösningar genom en modell liknande den som tidigare användes för bakterieexperiment. Efter belysning (eller förvaring i kärlet för de negativa kontrollösningarna) skedde inkubation och utvärdering av tillväxt i suspensionerna vilket genomfördes vid upprepade tillfällen, dag 4 till 7 efter exponering. Tillväxten jämfördes därefter med respektive icke-belysta kontroll. Experimenten involverade endast en koncentration av riboflavin (0,01 %) men dosering av UVA var i enlighet med tidigare experiment klinisk dos samt dubbla denna, för att kunna utvärdera ett dos-respons förhållande gällande ultraviolett ljus. Tillväxten hämmades av ultraviolett ljus med en reduktion av patogener över tid. Tillsats av riboflavin visade ingen tendens till ökad avdödning av mikroorganismer.

Studierna som genomförts i denna avhandling talar starkt för att fotoaktiveringen av riboflavin som idag tillämpas i CXL har potential att eliminera olika mikroorganismer och att terapin kan inducera läkning av mikrobiell keratit. Svårbehandlade kliniska infektiösa hornhinnesår har kunnat hanteras och i genomförd pilotstudie läkte majoriteten av infektionerna utan behov av antibiotika. Baserat på resultaten från dessa studier torde man kunna överväga CXL som tilläggsbehandling i svåra fall av mikrobiell keratit, nedsmältning av hornhinnan samt i fall då progress trots given antibiotikabehandling skett. Innan tillämpning som behandlingen som primär terapi eller i mer standardiserat förfarande måste denna utvärderas ytterligare i större och kliniska randomiserade studier. En implementering av fotokemisk riboflavin-UVA behandling skulle kunna innebära stora vinster i reducering av inneliggande vård, minskat antal komplikationer i samband med tillståndet samt en potentiell möjlighet att behandla tillstånd där idag endast bristfällig terapi finns att tillgå.


Acknowledgements I have been truly blessed in multiple ways, one of which is to be involved in a project that is highly fascinating and interesting to me as well as several of my colleagues and collaborators. The work presented here would not have been attainable without the help and assistance of a number of individuals, to whom I wish to convey my deepest gratitude. Sven Crafoord, my supervisor, has introduced me into the world of medical research with an ever-positive attitude. His support, guidance, and capacity to simplify every detail around the thesis work have facilitated the progression of the work immensely. Also, observing that it is possible combining the qualities of being a skilled surgeon with that of a researcher has been a true inspiration. Jes Mortensen, my clinical mentor during my Ophthalmological training and the Principal Investigator in the pilot study (Paper III), has to our knowledge been the first to implement CXL as an infectious therapy in Scandinavia. His energy, initiative, and generosity have been the core of a collaboration and friendship which hopefully will continue in further research investigations. Anders Bäckman, my co-supervisor, who has been the central character in all the experiments and of great help in developing the models for microbial elimination. His contribution signifies an imperative effort regarding the work, whose expertise and accessibility for discussions or questions are truly appreciated. Lennart Löfgren, my second co-supervisor, who helped us to get started with scientific discussions and planning of investigational models. Anders Magnuson, for educating and supporting me in the field of medical statistics. Guy Shanks for being a source of motivation, regarding theoretical as well as surgical ophthalmological abilities. Furthermore, for diligently supervising my introduction in vitreoretinal surgery. Dan Öhman, for encouraging me to start with this project and for, in several ways, supporting my development in research as well as surgery.


Petra Hedlund, for facilitating a number of practical and administrative issues around the research work and Niklas Karlsson, for Excel support. Patrick Lassarén, who meticulously guided my first steps on the path of Ophthalmic surgery. Ingrid Johansson who, as former Clinic Chief, employed me and at an early stage encouraged me to take on my first research project. Eva Karlsson, current Head of the Department of Ophthalmology, for allowing me to combine clinical development with time for research, and who genuinely tries to keep our working atmosphere positive. Inger Dedorsson and Helena Sönne, both for promoting the progression of our work. All the colleagues and co-workers at the Department of Opthalmology, Örebro University Hospital (USÖ), for making it a stimulating environment and a clinic with high aims regarding quality of health care. The studies involved here have been realizable through grants provided by the County Council (Forskningskommittén) and the experiments have been conducted at the Clinical Research Centre (KFC), USÖ. Finally, I wish to express my gratitude and love for my entire family. Gerd, my mother, who at first gave me a true thirst for knowledge, for her patience and constant encouragement, since as long I can remember. Mohamed, my father, for being a true example in so many aspects of life and whose attitude of never letting any obstacle hinder you, I admire. Omar, my brother, for, in every sense of the word, being just that. Romana, my wife, for keeping up with me, sharing her life with me, and persistently believing in me, many times more than myself. Muna and Makeen, for their love.


References 1. Wollensak G, Sporl E, Seiler T. [Treatment of keratoconus by

collagen cross linking]. Ophthalmologe 2003;100(1):44-9. 2. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-a-induced

collagen crosslinking for the treatment of keratoconus. Am J Ophthalmol 2003;135(5):620-7.

3. Wollensak G. Crosslinking treatment of progressive keratoconus: new hope. Curr Opin Ophthalmol 2006;17(4):356-60.

4. Wittig-Silva C, Whiting M, Lamoureux E, Lindsay RG, Sullivan LJ, Snibson GR. A randomized controlled trial of corneal collagen cross-linking in progressive keratoconus: preliminary results. J Refract Surg 2008;24(7):S720-5.

5. Hersh PS, Greenstein SA, Fry KL. Corneal collagen crosslinking for keratoconus and corneal ectasia: One-year results. J Cataract Refract Surg 2011;37(1):149-60.

6. Caporossi A, Mazzotta C, Baiocchi S, Caporossi T. Long-term results of riboflavin ultraviolet a corneal collagen cross-linking for keratoconus in Italy: the Siena eye cross study. Am J Ophthalmol 2010;149(4):585-93.

7. Coskunseven E, Jankov MR, 2nd, Hafezi F. Contralateral eye study of corneal collagen cross-linking with riboflavin and UVA irradiation in patients with keratoconus. J Refract Surg 2009;25(4):371-6.

8. Raiskup-Wolf F, Hoyer A, Spoerl E, Pillunat LE. Collagen crosslinking with riboflavin and ultraviolet-A light in keratoconus: long-term results. J Cataract Refract Surg 2008;34(5):796-801.

9. Hafezi F, Kanellopoulos J, Wiltfang R, Seiler T. Corneal collagen crosslinking with riboflavin and ultraviolet A to treat induced keratectasia after laser in situ keratomileusis. J Cataract Refract Surg 2007;33(12):2035-40.

10. Salgado JP, Khoramnia R, Lohmann CP, Winkler von Mohrenfels C. Corneal collagen crosslinking in post-LASIK keratectasia. Br J Ophthalmol 2011;95(4):493-7.

11. Kanellopoulos AJ, Binder PS. Management of corneal ectasia after LASIK with combined, same-day, topography-guided partial transepithelial PRK and collagen cross-linking: the athens protocol. J Refract Surg 2011;27(5):323-31.

12. Stojanovic A, Zhang J, Chen X, Nitter TA, Chen S, Wang Q. Topography-guided transepithelial surface ablation followed by corneal collagen cross-linking performed in a single combined procedure for the treatment of keratoconus and pellucid marginal degeneration. J Refract Surg 2010;26(2):145-52.


13. Coskunseven E, Jankov MR, 2nd, Hafezi F, Atun S, Arslan E, Kymionis GD. Effect of treatment sequence in combined intrastromal corneal rings and corneal collagen crosslinking for keratoconus. J Cataract Refract Surg 2009;35(12):2084-91.

14. El-Raggal TM. Effect of corneal collagen crosslinking on femtosecond laser channel creation for intrastromal corneal ring segment implantation in keratoconus. J Cataract Refract Surg 2011;37(4):701-5.

15. Kanellopoulos AJ. Comparison of sequential vs same-day simultaneous collagen cross-linking and topography-guided PRK for treatment of keratoconus. J Refract Surg 2009;25(9):S812-8.

16. Sporl E, Huhle M, Kasper M, Seiler T. [Increased rigidity of the cornea caused by intrastromal cross-linking]. Ophthalmologe 1997;94(12):902-6.

17. Spoerl E, Huhle M, Seiler T. Induction of cross-links in corneal tissue. Exp Eye Res 1998;66(1):97-103.

18. Spoerl E, Seiler T. Techniques for stiffening the cornea. J Refract Surg 1999;15(6):711-3.

19. Wollensak G, Spoerl E, Seiler T. Stress-strain measurements of human and porcine corneas after riboflavin-ultraviolet-A-induced cross-linking. J Cataract Refract Surg 2003;29(9):1780-5.

20. Spoerl E, Wollensak G, Dittert DD, Seiler T. Thermomechanical behavior of collagen-cross-linked porcine cornea. Ophthalmologica 2004;218(2):136-40.

21. Spoerl E, Wollensak G, Seiler T. Increased resistance of crosslinked cornea against enzymatic digestion. Curr Eye Res 2004;29(1):35-40.

22. Dhaliwal JS, Kaufman SC. Corneal collagen cross-linking: a confocal, electron, and light microscopy study of eye bank corneas. Cornea 2009;28(1):62-7.

23. Bottos KM, Dreyfuss JL, Regatieri CV, Lima-Filho AA, Schor P, Nader HB, Chamon W. Immunofluorescence confocal microscopy of porcine corneas following collagen cross-linking treatment with riboflavin and ultraviolet A. J Refract Surg 2008;24(7):S715-9.

24. Wollensak G, Spoerl E, Wilsch M, Seiler T. Keratocyte apoptosis after corneal collagen cross-linking using riboflavin/UVA treatment. Cornea 2004;23(1):43-9.

25. Wollensak G, Sporl E, Reber F, Pillunat L, Funk R. Corneal endothelial cytotoxicity of riboflavin/UVA treatment in vitro. Ophthalmic Res 2003;35(6):324-8.

26. Wollensak G, Spoerl E, Wilsch M, Seiler T. Endothelial cell damage after riboflavin-ultraviolet-A treatment in the rabbit. J Cataract Refract Surg 2003;29(9):1786-90.

27. Wollensak G, Iomdina E, Dittert DD, Herbst H. Wound healing in the rabbit cornea after corneal collagen cross-linking with riboflavin and UVA. Cornea 2007;26(5):600-5.


28. Kymionis GD, Diakonis VF, Kalyvianaki M, Portaliou D, Siganos C, Kozobolis VP, Pallikaris AI. One-year follow-up of corneal confocal microscopy after corneal cross-linking in patients with post laser in situ keratosmileusis ectasia and keratoconus. Am J Ophthalmol 2009;147(5):774-8, 8 e1.

29. Doors M, Tahzib NG, Eggink FA, Berendschot TT, Webers CA, Nuijts RM. Use of anterior segment optical coherence tomography to study corneal changes after collagen cross-linking. Am J Ophthalmol 2009;148(6):844-51 e2.

30. Hafezi F, Mrochen M, Iseli HP, Seiler T. Collagen crosslinking with ultraviolet-A and hypoosmolar riboflavin solution in thin corneas. J Cataract Refract Surg 2009;35(4):621-4.

31. Boxer Wachler BS, Pinelli R, Ertan A, Chan CC. Safety and efficacy of transepithelial crosslinking (C3-R/CXL). J Cataract Refract Surg 2010;36(1):186-8; author reply 8-9.

32. Baiocchi S, Mazzotta C, Cerretani D, Caporossi T, Caporossi A. Corneal crosslinking: riboflavin concentration in corneal stroma exposed with and without epithelium. J Cataract Refract Surg 2009;35(5):893-9.

33. Samaras K, O'Brart D P, Doutch J, Hayes S, Marshall J, Meek KM. Effect of epithelial retention and removal on riboflavin absorption in porcine corneas. J Refract Surg 2009;25(9):771-5.

34. Hayes S, O'Brart DP, Lamdin LS, Doutch J, Samaras K, Marshall J, Meek KM. Effect of complete epithelial debridement before riboflavin-ultraviolet-A corneal collagen crosslinking therapy. J Cataract Refract Surg 2008;34(4):657-61.

35. Wollensak G, Iomdina E. Biomechanical and histological changes after corneal crosslinking with and without epithelial debridement. J Cataract Refract Surg 2009;35(3):540-6.

36. Greenstein SA, Fry KL, Hersh PS. Corneal topography indices after corneal collagen crosslinking for keratoconus and corneal ectasia: one-year results. J Cataract Refract Surg 2011;37(7):1282-90.

37. Greenstein SA, Fry KL, Bhatt J, Hersh PS. Natural history of corneal haze after collagen crosslinking for keratoconus and corneal ectasia: Scheimpflug and biomicroscopic analysis. J Cataract Refract Surg 2010;36(12):2105-14.

38. Raiskup F, Hoyer A, Spoerl E. Permanent corneal haze after riboflavin-UVA-induced cross-linking in keratoconus. J Refract Surg 2009;25(9):S824-8.

39. Mazzotta C, Balestrazzi A, Baiocchi S, Traversi C, Caporossi A. Stromal haze after combined riboflavin-UVA corneal collagen cross-linking in keratoconus: in vivo confocal microscopic evaluation. Clin Experiment Ophthalmol 2007;35(6):580-2.


40. Gokhale NS, Vemuganti GK. Diclofenac-induced acute corneal melt after collagen crosslinking for keratoconus. Cornea 2010;29(1):117-9.

41. Labiris G, Kaloghianni E, Koukoula S, Zissimopoulos A, Kozobolis VP. Corneal melting after collagen cross-linking for keratoconus: a case report. J Med Case Reports 2011;5:152.

42. Faschinger C, Kleinert R, Wedrich A. [Corneal melting in both eyes after simultaneous corneal cross-linking in a patient with keratoconus and Down syndrome]. Ophthalmologe 2010;107(10):951-2, 4-5.

43. Sharma N, Maharana P, Singh G, Titiyal JS. Pseudomonas keratitis after collagen crosslinking for keratoconus: case report and review of literature. J Cataract Refract Surg 2010;36(3):517-20.

44. Perez-Santonja JJ, Artola A, Javaloy J, Alio JL, Abad JL. Microbial keratitis after corneal collagen crosslinking. J Cataract Refract Surg 2009;35(6):1138-40.

45. Rama P, Di Matteo F, Matuska S, Paganoni G, Spinelli A. Acanthamoeba keratitis with perforation after corneal crosslinking and bandage contact lens use. J Cataract Refract Surg 2009;35(4):788-91.

46. Pollhammer M, Cursiefen C. Bacterial keratitis early after corneal crosslinking with riboflavin and ultraviolet-A. J Cataract Refract Surg 2009;35(3):588-9.

47. Kymionis GD, Portaliou DM, Bouzoukis DI, Suh LH, Pallikaris AI, Markomanolakis M, Yoo SH. Herpetic keratitis with iritis after corneal crosslinking with riboflavin and ultraviolet A for keratoconus. J Cataract Refract Surg 2007;33(11):1982-4.

48. Kymionis GD, Bouzoukis DI, Diakonis VF, Portaliou DM, Pallikaris AI, Yoo SH. Diffuse lamellar keratitis after corneal crosslinking in a patient with post-laser in situ keratomileusis corneal ectasia. J Cataract Refract Surg 2007;33(12):2135-7.

49. Mazzotta C, Balestrazzi A, Traversi C, Baiocchi S, Caporossi T, Tommasi C, Caporossi A. Treatment of progressive keratoconus by riboflavin-UVA-induced cross-linking of corneal collagen: ultrastructural analysis by Heidelberg Retinal Tomograph II in vivo confocal microscopy in humans. Cornea 2007;26(4):390-7.

50. Goldich Y, Marcovich AL, Barkana Y, Avni I, Zadok D. Safety of corneal collagen cross-linking with UV-A and riboflavin in progressive keratoconus. Cornea 2010;29(4):409-11.

51. Mazzotta C, Traversi C, Baiocchi S, Caporossi O, Bovone C, Sparano MC, Balestrazzi A, Caporossi A. Corneal healing after riboflavin ultraviolet-A collagen cross-linking determined by confocal laser scanning microscopy in vivo: early and late modifications. Am J Ophthalmol 2008;146(4):527-33.


52. Mazzotta C, Traversi C, Baiocchi S, Sergio P, Caporossi T, Caporossi A. Conservative treatment of keratoconus by riboflavin-uva-induced cross-linking of corneal collagen: qualitative investigation. Eur J Ophthalmol 2006;16(4):530-5.

53. Vinciguerra P, Albe E, Trazza S, Seiler T, Epstein D. Intraoperative and postoperative effects of corneal collagen cross-linking on progressive keratoconus. Arch Ophthalmol 2009;127(10):1258-65.

54. Vinciguerra P, Albe E, Trazza S, Rosetta P, Vinciguerra R, Seiler T, Epstein D. Refractive, topographic, tomographic, and aberrometric analysis of keratoconic eyes undergoing corneal cross-linking. Ophthalmology 2009;116(3):369-78.

55. Koller T, Mrochen M, Seiler T. Complication and failure rates after corneal crosslinking. J Cataract Refract Surg 2009;35(8):1358-62.

56. Keating A, Pineda R, 2nd, Colby K. Corneal cross linking for keratoconus. Semin Ophthalmol 2010;25(5-6):249-55.

57. Ashwin PT, McDonnell PJ. Collagen cross-linkage: a comprehensive review and directions for future research. Br J Ophthalmol 2010;94(8):965-70.

58. Kolli S, Aslanides IM. Safety and efficacy of collagen crosslinking for the treatment of keratoconus. Expert Opin Drug Saf 2010;9(6):949-57.

59. Powers HJ. Riboflavin (vitamin B-2) and health. Am J Clin Nutr 2003;77(6):1352-60.

60. Food Standards Agency, U.K. [cited 6 Oct 2011]. Available from: http://www.food.gov.uk/safereating/chemsafe/additivesbranch/enumberlist.

61. Kurtin WE, Song PS. Photochemistry of the model phototropic system involving flavins and indoles. I. Fluorescence polarization and MO calculations of the direction of the electronic transition moments in flavins. Photochem Photobiol 1968;7(3):263-73.

62. Wolken JJ. Microspectrophotometry and the photoreceptor of Phycomyces I. J Cell Biol 1969;43(2):354-60.

63. Cairns WL, Metzler DE. Photochemical degradation of flavins. VI. A new photoproduct and its use in studying the photolytic mechanism. J Am Chem Soc 1971;93(11):2772-7.

64. Hardwick CC, Herivel TR, Hernandez SC, Ruane PH, Goodrich RP. Separation, identification and quantification of riboflavin and its photoproducts in blood products using high-performance liquid chromatography with fluorescence detection: a method to support pathogen reduction technology. Photochem Photobiol 2004;80(3):609-15.

65. Baier J, Maisch T, Maier M, Engel E, Landthaler M, Baumler W. Singlet oxygen generation by UVA light exposure of endogenous photosensitizers. Biophys J 2006;91(4):1452-9.


66. Joshi PC. Ultraviolet radiation-induced photodegradation and 1O2, O2-. production by riboflavin, lumichrome and lumiflavin. Indian J Biochem Biophys 1989;26(3):186-9.

67. Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, Moan J, Peng Q. Photodynamic therapy. J Natl Cancer Inst 1998;90(12):889-905.

68. Nowak-Sliwinska P, Karocki A, Elas M, Pawlak A, Stochel G, Urbanska K. Verteporfin, photofrin II, and merocyanine 540 as PDT photosensitizers against melanoma cells. Biochem Biophys Res Commun 2006;349(2):549-55.

69. Nowis D, Makowski M, Stoklosa T, Legat M, Issat T, Golab J. Direct tumor damage mechanisms of photodynamic therapy. Acta Biochim Pol 2005;52(2):339-52.

70. Kim H, Kirschenbaum LJ, Rosenthal I, Riesz P. Photosensitized formation of ascorbate radicals by riboflavin: an ESR study. Photochem Photobiol 1993;57(5):777-84.

71. Kumari MV, Yoneda T, Hiramatsu M. Scavenging activity of "beta catechin" on reactive oxygen species generated by photosensitization of riboflavin. Biochem Mol Biol Int 1996;38(6):1163-70.

72. Sato K, Taguchi H, Maeda T, Minami H, Asada Y, Watanabe Y, Yoshikawa K. The primary cytotoxicity in ultraviolet-a-irradiated riboflavin solution is derived from hydrogen peroxide. J Invest Dermatol 1995;105(4):608-12.

73. Minami H, Sato K, Maeda T, Taguchi H, Yoshikawa K, Kosaka H, Shiga T, Tsuji T. Hypoxia potentiates ultraviolet A-induced riboflavin cytotoxicity. J Invest Dermatol 1999;113(1):77-81.

74. Tsugita A, Okada Y, Uehara K. Photosensitized inactivation of ribonucleic acids in the presence of riboflavin. Biochim Biophys Acta 1965;103(2):360-3.

75. Ito K, Inoue S, Yamamoto K, Kawanishi S. 8-Hydroxydeoxyguanosine formation at the 5' site of 5'-GG-3' sequences in double-stranded DNA by UV radiation with riboflavin. J Biol Chem 1993;268(18):13221-7.

76. Dalle Carbonare M, Pathak MA. Skin photosensitizing agents and the role of reactive oxygen species in photoaging. J Photochem Photobiol B 1992;14(1-2):105-24.

77. Mahns A, Melchheier I, Suschek CV, Sies H, Klotz LO. Irradiation of cells with ultraviolet-A (320-400 nm) in the presence of cell culture medium elicits biological effects due to extracellular generation of hydrogen peroxide. Free Radic Res 2003;37(4):391-7.

78. Corbin F, 3rd. Pathogen inactivation of blood components: current status and introduction of an approach using riboflavin as a photosensitizer. Int J Hematol 2002;76 Suppl 2:253-7.


79. Olsen JH, Hertz H, Kjaer SK, Bautz A, Mellemkjaer L, Boice JD, Jr. Childhood leukemia following phototherapy for neonatal hyperbilirubinemia (Denmark). Cancer Causes Control 1996;7(4):411-4.

80. Sisson TR. Photodegradation of riboflavin in neonates. Fed Proc 1987;46(5):1883-5.

81. Gromisch DS, Lopez R, Cole HS, Cooperman JM. Light (phototherapy)--induced riboflavin deficiency in the neonate. J Pediatr 1977;90(1):118-22.

82. Hovi L, Hekali R, Siimes MA. Evidence of riboflavin depletion in breast-fed newborns and its further acceleration during treatment of hyperbilirubinemia by phototherapy. Acta Paediatr Scand 1979;68(4):567-70.

83. Ruane PH, Edrich R, Gampp D, Keil SD, Leonard RL, Goodrich RP. Photochemical inactivation of selected viruses and bacteria in platelet concentrates using riboflavin and light. Transfusion (Paris) 2004;44(6):877-85.

84. Cardo LJ, Rentas FJ, Ketchum L, Salata J, Harman R, Melvin W, Weina PJ, Mendez J, Reddy H, Goodrich R. Pathogen inactivation of Leishmania donovani infantum in plasma and platelet concentrates using riboflavin and ultraviolet light. Vox Sang 2006;90(2):85-91.

85. Cardo LJ, Salata J, Mendez J, Reddy H, Goodrich R. Pathogen inactivation of Trypanosoma cruzi in plasma and platelet concentrates using riboflavin and ultraviolet light. Transfus Apher Sci 2007;37(2):131-7.

86. Tonnetti L, Proctor MC, Reddy HL, Goodrich RP, Leiby DA. Evaluation of the Mirasol pathogen [corrected] reduction technology system against Babesia microti in apheresis platelets and plasma. Transfusion (Paris) 2010;50(5):1019-27.

87. Goodrich RP. The use of riboflavin for the inactivation of pathogens in blood products. Vox Sang 2000;78 Suppl 2:211-5.

88. Perez-Pujol S, Tonda R, Lozano M, Fuste B, Lopez-Vilchez I, Galan AM, Li J, Goodrich R, Escolar G. Effects of a new pathogen-reduction technology (Mirasol PRT) on functional aspects of platelet concentrates. Transfusion (Paris) 2005;45(6):911-9.

89. Li J, Lockerbie O, de Korte D, Rice J, McLean R, Goodrich RP. Evaluation of platelet mitochondria integrity after treatment with Mirasol pathogen reduction technology. Transfusion (Paris) 2005;45(6):920-6.

90. Goodrich RP, Edrich RA, Li J, Seghatchian J. The Mirasol PRT system for pathogen reduction of platelets and plasma: an overview of current status and future trends. Transfus Apher Sci 2006;35(1):5-17.


91. Marschner S, Goodrich R. Pathogen Reduction Technology Treatment of Platelets, Plasma and Whole Blood Using Riboflavin and UV Light. Transfus Med Hemother 2011;38(1):8-18.

92. Pelletier JP, Transue S, Snyder EL. Pathogen inactivation techniques. Best Pract Res Clin Haematol 2006;19(1):205-42.

93. Seghatchian J, de Sousa G. Pathogen-reduction systems for blood components: the current position and future trends. Transfus Apher Sci 2006;35(3):189-96.

94. Solheim BG. Pathogen reduction of blood components. Transfus Apher Sci 2008;39(1):75-82.

95. Politis C, Kavallierou L, Hantziara S, Katsea P, Triantaphylou V, Richardson C, Tsoutsos D, Anagnostopoulos N, Gorgolidis G, Ziroyannis P. Quality and safety of fresh-frozen plasma inactivated and leucoreduced with the Theraflex methylene blue system including the Blueflex filter: 5 years' experience. Vox Sang 2007;92(4):319-26.

96. Seghatchian J, Walker WH, Reichenberg S. Updates on pathogen inactivation of plasma using Theraflex methylene blue system. Transfus Apher Sci 2008;38(3):271-80.

97. Janetzko K, Lin L, Eichler H, Mayaudon V, Flament J, Kluter H. Implementation of the INTERCEPT Blood System for Platelets into routine blood bank manufacturing procedures: evaluation of apheresis platelets. Vox Sang 2004;86(4):239-45.

98. Irsch J, Lin L. Pathogen Inactivation of Platelet and Plasma Blood Components for Transfusion Using the INTERCEPT Blood System. Transfus Med Hemother 2011;38(1):19-31.

99. Irsch J, Pinkoski L, Corash L, Lin L. INTERCEPT plasma: comparability with conventional fresh-frozen plasma based on coagulation function--an in vitro analysis. Vox Sang 2010;98(1):47-55.

100. Galan AM, Lozano M, Molina P, Navalon F, Marschner S, Goodrich R, Escolar G. Impact of pathogen reduction technology and storage in platelet additive solutions on platelet function. Transfusion (Paris) 2011;51(4):808-15.

101. A randomized controlled clinical trial evaluating the performance and safety of platelets treated with MIRASOL pathogen reduction technology. Transfusion (Paris) 2010;50(11):2362-75.

102. Ettinger A, Miklauz MM, Hendrix BK, Bihm DJ, Maldonado-Codina G, Goodrich RP. Protein stability of previously frozen plasma, riboflavin and UV light-treated, refrozen and stored for up to 2 years at -30 degrees C. Transfus Apher Sci 2011;44(1):25-31.

103. Fast LD, DiLeone G, Marschner S. Inactivation of human white blood cells in platelet products after pathogen reduction technology treatment in comparison to gamma irradiation. Transfusion (Paris) 2011;51(7):1397-404.


104. Fast LD, Dileone G, Li J, Goodrich R. Functional inactivation of white blood cells by Mirasol treatment. Transfusion (Paris) 2006;46(4):642-8.

105. Reddy HL, Dayan AD, Cavagnaro J, Gad S, Li J, Goodrich RP. Toxicity testing of a novel riboflavin-based technology for pathogen reduction and white blood cell inactivation. Transfus Med Rev 2008;22(2):133-53.

106. Fast LD, DiLeone G, Cardarelli G, Li J, Goodrich R. Mirasol PRT treatment of donor white blood cells prevents the development of xenogeneic graft-versus-host disease in Rag2-/-gamma c-/- double knockout mice. Transfusion (Paris) 2006;46(9):1553-60.

107. Marschner S, Fast LD, Baldwin WM, 3rd, Slichter SJ, Goodrich RP. White blood cell inactivation after treatment with riboflavin and ultraviolet light. Transfusion (Paris) 2010;50(11):2489-98.

108. Keay L, Edwards K, Naduvilath T, Taylor HR, Snibson GR, Forde K, Stapleton F. Microbial keratitis predisposing factors and morbidity. Ophthalmology 2006;113(1):109-16.

109. Bourcier T, Thomas F, Borderie V, Chaumeil C, Laroche L. Bacterial keratitis: predisposing factors, clinical and microbiological review of 300 cases. Br J Ophthalmol 2003;87(7):834-8.

110. Saeed A, D'Arcy F, Stack J, Collum LM, Power W, Beatty S. Risk factors, microbiological findings, and clinical outcomes in cases of microbial keratitis admitted to a tertiary referral center in ireland. Cornea 2009;28(3):285-92.

111. Green M, Apel A, Stapleton F. Risk factors and causative organisms in microbial keratitis. Cornea 2008;27(1):22-7.

112. Whitcher JP, Srinivasan M. Corneal ulceration in the developing world--a silent epidemic. Br J Ophthalmol 1997;81(8):622-3.

113. Thylefors B. Present challenges in the global prevention of blindness. Aust N Z J Ophthalmol 1992;20(2):89-94.

114. Jeng BH, Gritz DC, Kumar AB, Holsclaw DS, Porco TC, Smith SD, Whitcher JP, Margolis TP, Wong IG. Epidemiology of ulcerative keratitis in Northern California. Arch Ophthalmol 2010;128(8):1022-8.

115. Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2000;2(9):1051-60.

116. Fleiszig SM, Evans DJ. The pathogenesis of bacterial keratitis: studies with Pseudomonas aeruginosa. Clin Exp Optom 2002;85(5):271-8.

117. Vasil ML. Pseudomonas aeruginosa: biology, mechanisms of virulence, epidemiology. J Pediatr 1986;108(5 Pt 2):800-5.

118. Constantinou M, Jhanji V, Tao LW, Vajpayee RB. Clinical review of corneal ulcers resulting in evisceration and enucleation in elderly


population. Graefes Arch Clin Exp Ophthalmol 2009;247(10):1389-93.

119. Cruz CS, Cohen EJ, Rapuano CJ, Laibson PR. Microbial keratitis resulting in loss of the eye. Ophthalmic Surg Lasers 1998;29(10):803-7.

120. Scott IU, Flynn HW, Jr., Feuer W, Pflugfelder SC, Alfonso EC, Forster RK, Miller D. Endophthalmitis associated with microbial keratitis. Ophthalmology 1996;103(11):1864-70.

121. Titiyal JS, Negi S, Anand A, Tandon R, Sharma N, Vajpayee RB. Risk factors for perforation in microbial corneal ulcers in north India. Br J Ophthalmol 2006;90(6):686-9.

122. Mutoh T, Ishikawa I, Matsumoto Y, Chikuda M. A retrospective study of nine cases of Acanthamoeba keratitis. Clin Ophthalmol 2010;4:1189-92.

123. Tu EY, Joslin CE. Recent outbreaks of atypical contact lens-related keratitis: what have we learned? Am J Ophthalmol 2010;150(5):602-8 e2.

124. Tanhehco T, Colby K. The clinical experience of Acanthamoeba keratitis at a tertiary care eye hospital. Cornea 2010;29(9):1005-10.

125. Panjwani N. Pathogenesis of acanthamoeba keratitis. Ocul Surf 2010;8(2):70-9.

126. Qian Y, Meisler DM, Langston RH, Jeng BH. Clinical experience with Acanthamoeba keratitis at the cole eye institute, 1999-2008. Cornea 2010;29(9):1016-21.

127. Oldenburg CE, Keenan JD, Cevallos V, Chan MF, Acharya NR, Gaynor BD, McLeod SD, Esterberg EJ, Porco TC, Lietman TM. Microbiological cure times in acanthamoeba keratitis. Eye (Lond) 2011;25(9):1155-60.

128. Rahman MR, Johnson GJ, Husain R, Howlader SA, Minassian DC. Randomised trial of 0.2% chlorhexidine gluconate and 2.5% natamycin for fungal keratitis in Bangladesh. Br J Ophthalmol 1998;82(8):919-25.

129. Thomas PA, Leck AK, Myatt M. Characteristic clinical features as an aid to the diagnosis of suppurative keratitis caused by filamentous fungi. Br J Ophthalmol 2005;89(12):1554-8.

130. Florcruz NV, Peczon I, Jr. Medical interventions for fungal keratitis. Cochrane Database Syst Rev 2008(1):CD004241.

131. Shen YC, Wang CY, Tsai HY, Lee HN. Intracameral voriconazole injection in the treatment of fungal endophthalmitis resulting from keratitis. Am J Ophthalmol 2010;149(6):916-21.

132. Bhartiya P, Daniell M, Constantinou M, Islam FM, Taylor HR. Fungal keratitis in Melbourne. Clin Experiment Ophthalmol 2007;35(2):124-30.


133. Tanure MA, Cohen EJ, Sudesh S, Rapuano CJ, Laibson PR. Spectrum of fungal keratitis at Wills Eye Hospital, Philadelphia, Pennsylvania. Cornea 2000;19(3):307-12.

134. Boyce JM. Consequences of inaction: importance of infection control practices. Clin Infect Dis 2001;33 Suppl 3:S133-7.

135. Holmberg SD, Solomon SL, Blake PA. Health and economic impacts of antimicrobial resistance. Rev Infect Dis 1987;9(6):1065-78.

136. Cosgrove SE. The relationship between antimicrobial resistance and patient outcomes: mortality, length of hospital stay, and health care costs. Clin Infect Dis 2006;42 Suppl 2:S82-9.

137. Rejiba S, Limam F, Belhadj C, Belhadj O, Ben-Mahrez K. Biochemical characterization of a novel extended-spectrum beta-lactamase from Pseudomonas aeruginosa 802. Microb Drug Resist 2002;8(1):9-13.

138. Philippon LN, Naas T, Bouthors AT, Barakett V, Nordmann P. OXA-18, a class D clavulanic acid-inhibited extended-spectrum beta-lactamase from Pseudomonas aeruginosa. Antimicrob Agents Chemother 1997;41(10):2188-95.

139. Johnson AP. Methicillin-resistant Staphylococcus aureus: the European landscape. J Antimicrob Chemother 2011;66 Suppl 4:iv43-iv8.

140. Falagas ME, Bliziotis IA. Pandrug-resistant Gram-negative bacteria: the dawn of the post-antibiotic era? Int J Antimicrob Agents 2007;29(6):630-6.

141. Souli M, Galani I, Giamarellou H. Emergence of extensively drug-resistant and pandrug-resistant Gram-negative bacilli in Europe. Euro Surveill 2008;13(47).

142. Manchanda V, Sanchaita S, Singh N. Multidrug resistant acinetobacter. J Glob Infect Dis 2010;2(3):291-304.

143. Jayakumar S, Appalaraju B. Prevalence of multi and pan drug resistant Pseudomonas aeruginosa with respect to ESBL and MBL in a tertiary care hospital. Indian J Pathol Microbiol 2007;50(4):922-5.

144. Chalita MR, Hofling-Lima AL, Paranhos A, Jr., Schor P, Belfort R, Jr. Shifting trends in in vitro antibiotic susceptibilities for common ocular isolates during a period of 15 years. Am J Ophthalmol 2004;137(1):43-51.

145. Alexandrakis G, Alfonso EC, Miller D. Shifting trends in bacterial keratitis in south Florida and emerging resistance to fluoroquinolones. Ophthalmology 2000;107(8):1497-502.

146. Green M, Apel A, Stapleton F. A longitudinal study of trends in keratitis in Australia. Cornea 2008;27(1):33-9.


147. Shalchi Z, Gurbaxani A, Baker M, Nash J. Antibiotic Resistance in Microbial Keratitis: Ten-Year Experience of Corneal Scrapes in the United Kingdom. Ophthalmology 2011.

148. Tuft SJ, Matheson M. In vitro antibiotic resistance in bacterial keratitis in London. Br J Ophthalmol 2000;84(7):687-91.

149. Orlans HO, Hornby SJ, Bowler IC. In vitro antibiotic susceptibility patterns of bacterial keratitis isolates in Oxford, UK: a 10-year review. Eye (Lond) 2011;25(4):489-93.

150. Sueke H, Kaye S, Neal T, Murphy C, Hall A, Whittaker D, Tuft S, Parry C. Minimum inhibitory concentrations of standard and novel antimicrobials for isolates from bacterial keratitis. Invest Ophthalmol Vis Sci 2010;51(5):2519-24.

151. Afshari NA, Ma JJ, Duncan SM, Pineda R, Starr CE, Decroos FC, Johnson CS, Adelman RA. Trends in resistance to ciprofloxacin, cefazolin, and gentamicin in the treatment of bacterial keratitis. J Ocul Pharmacol Ther 2008;24(2):217-23.

152. Scoper SV. Review of third-and fourth-generation fluoroquinolones in ophthalmology: in-vitro and in-vivo efficacy. Adv Ther 2008;25(10):979-94.

153. Bertino JS. Impact of antibiotic resistance in the management of ocular infections: the role of current and future antibiotics. Clin Ophthalmol 2009;3:507-21.

154. Haas W, Pillar CM, Hesje CK, Sanfilippo CM, Morris TW. Bactericidal activity of besifloxacin against staphylococci, Streptococcus pneumoniae and Haemophilus influenzae. J Antimicrob Chemother 2010;65(7):1441-7.

155. Sanders ME, Moore QC, 3rd, Norcross EW, Sanfilippo CM, Hesje CK, Shafiee A, Marquart ME. Comparison of besifloxacin, gatifloxacin, and moxifloxacin against strains of pseudomonas aeruginosa with different quinolone susceptibility patterns in a rabbit model of keratitis. Cornea 2011;30(1):83-90.

156. Jayahar Bharathi M, Ramakrishnan R, Ramesh S, Murugan N. Extended-Spectrum Beta-Lactamase-Mediated Resistance among Bacterial Isolates Recovered from Ocular Infections. Ophthalmic Res 2011;47(1):52-6.

157. Reddy AK, Garg P, Babu KH, Gopinathan U, Sharma S. In vitro antibiotic susceptibility of rapidly growing nontuberculous mycobacteria isolated from patients with microbial keratitis. Curr Eye Res 2010;35(3):225-9.

158. Fintelmann RE, Hoskins EN, Lietman TM, Keenan JD, Gaynor BD, Cevallos V, Acharya NR. Topical fluoroquinolone use as a risk factor for in vitro fluoroquinolone resistance in ocular cultures. Arch Ophthalmol 2011;129(4):399-402.


159. Jhanji V, Sharma N, Satpathy G, Titiyal J. Fourth-generation fluoroquinolone-resistant bacterial keratitis. J Cataract Refract Surg 2007;33(8):1488-9.

160. Rufer F, Sander S, Klettner A, Frimpong-Boateng A, Erb C. Characterization of the thinnest point of the cornea compared with the central corneal thickness in normal subjects. Cornea 2009;28(2):177-80.

161. Khoramnia R, Rabsilber TM, Auffarth GU. Central and peripheral pachymetry measurements according to age using the Pentacam rotating Scheimpflug camera. J Cataract Refract Surg 2007;33(5):830-6.

162. Prasad A, Fry K, Hersh PS. Relationship of age and refraction to central corneal thickness. Cornea 2011;30(5):553-5.

163. Ehlers N, Hjortdal J. Corneal thickness: measurement and implications. Exp Eye Res 2004;78(3):543-8.

164. Ehlers N, Heegaard S, Hjortdal J, Ivarsen A, Nielsen K, Prause JU. Morphological evaluation of normal human corneal epithelium. Acta Ophthalmol 2010;88(8):858-61.

165. Patel S, McLaren J, Hodge D, Bourne W. Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo. Invest Ophthalmol Vis Sci 2001;42(2):333-9.

166. Pfister RR. The normal surface of corneal epithelium: a scanning electron microscopic study. Invest Ophthalmol 1973;12(9):654-68.

167. Lagali N, Germundsson J, Fagerholm P. The role of Bowman's layer in corneal regeneration after phototherapeutic keratectomy: a prospective study using in vivo confocal microscopy. Invest Ophthalmol Vis Sci 2009;50(9):4192-8.

168. Reinstein DZ, Archer TJ, Gobbe M, Silverman RH, Coleman DJ. Stromal thickness in the normal cornea: three-dimensional display with artemis very high-frequency digital ultrasound. J Refract Surg 2009;25(9):776-86.

169. Proulx S, d'Arc Uwamaliya J, Carrier P, Deschambeault A, Audet C, Giasson CJ, Guerin SL, Auger FA, Germain L. Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types. Mol Vis 2010;16:2192-201.

170. Komai Y, Ushiki T. The three-dimensional organization of collagen fibrils in the human cornea and sclera. Invest Ophthalmol Vis Sci 1991;32(8):2244-58.

171. DelMonte DW, Kim T. Anatomy and physiology of the cornea. J Cataract Refract Surg 2011;37(3):588-98.

172. Sivak JM, Fini ME. MMPs in the eye: emerging roles for matrix metalloproteinases in ocular physiology. Prog Retin Eye Res 2002;21(1):1-14.

173. Mustonen RK, McDonald MB, Srivannaboon S, Tan AL, Doubrava MW, Kim CK. Normal human corneal cell populations evaluated


by in vivo scanning slit confocal microscopy. Cornea 1998;17(5):485-92.

174. Hollingsworth J, Perez-Gomez I, Mutalib HA, Efron N. A population study of the normal cornea using an in vivo, slit-scanning confocal microscope. Optom Vis Sci 2001;78(10):706-11.

175. Peh GS, Beuerman RW, Colman A, Tan DT, Mehta JS. Human corneal endothelial cell expansion for corneal endothelium transplantation: an overview. Transplantation 2011;91(8):811-9.

176. Mathew JH, Goosey JD, Bergmanson JP. Quantified histopathology of the keratoconic cornea. Optom Vis Sci 2011;88(8):988-97.

177. Moller-Pedersen T. A comparative study of human corneal keratocyte and endothelial cell density during aging. Cornea 1997;16(3):333-8.

178. Niederer RL, Perumal D, Sherwin T, McGhee CN. Age-related differences in the normal human cornea: a laser scanning in vivo confocal microscopy study. Br J Ophthalmol 2007;91(9):1165-9.

179. Marfurt CF, Cox J, Deek S, Dvorscak L. Anatomy of the human corneal innervation. Exp Eye Res 2010;90(4):478-92.

180. Medawar PB. Immunity to homologous grafted skin; the fate of skin homografts transplanted to the brain, to subcutaneous tissue, and to the anterior chamber of the eye. Br J Exp Pathol 1948;29(1):58-69.

181. Hori J. Mechanisms of immune privilege in the anterior segment of the eye: what we learn from corneal transplantation. J Ocul Biol Dis Infor 2008;1(2-4):94-100.

182. Streilein JW. Ocular immune privilege: the eye takes a dim but practical view of immunity and inflammation. J Leukoc Biol 2003;74(2):179-85.

183. Hamrah P, Huq SO, Liu Y, Zhang Q, Dana MR. Corneal immunity is mediated by heterogeneous population of antigen-presenting cells. J Leukoc Biol 2003;74(2):172-8.

184. Knickelbein JE, Watkins SC, McMenamin PG, Hendricks RL. Stratification of Antigen-presenting Cells within the Normal Cornea. Ophthalmol Eye Dis 2009;1:45-54.

185. Kernacki KA, Barrett RP, Hobden JA, Hazlett LD. Macrophage inflammatory protein-2 is a mediator of polymorphonuclear neutrophil influx in ocular bacterial infection. J Immunol 2000;164(2):1037-45.

186. Rudner XL, Kernacki KA, Barrett RP, Hazlett LD. Prolonged elevation of IL-1 in Pseudomonas aeruginosa ocular infection regulates macrophage-inflammatory protein-2 production, polymorphonuclear neutrophil persistence, and corneal perforation. J Immunol 2000;164(12):6576-82.

187. Hazlett LD. Pathogenic mechanisms of P. aeruginosa keratitis: a review of the role of T cells, Langerhans cells, PMN, and cytokines. DNA Cell Biol 2002;21(5-6):383-90.


188. Callegan MC. Checks and balances: the ocular response to infection. Virulence 2010;1(4):222.

189. Steuhl KP, Doring G, Henni A, Thiel HJ, Botzenhart K. Relevance of host-derived and bacterial factors in Pseudomonas aeruginosa corneal infections. Invest Ophthalmol Vis Sci 1987;28(9):1559-68.

190. Hazlett LD, McClellan S, Kwon B, Barrett R. Increased severity of Pseudomonas aeruginosa corneal infection in strains of mice designated as Th1 versus Th2 responsive. Invest Ophthalmol Vis Sci 2000;41(3):805-10.

191. Clarke DW, Alizadeh H, Niederkorn JY. Intracorneal instillation of latex beads induces macrophage-dependent protection against Acanthamoeba keratitis. Invest Ophthalmol Vis Sci 2006;47(11):4917-25.

192. Avila MY, Navia JL. Effect of genipin collagen crosslinking on porcine corneas. J Cataract Refract Surg 2010;36(4):659-64.

193. Martins SA, Combs JC, Noguera G, Camacho W, Wittmann P, Walther R, Cano M, Dick J, Behrens A. Antimicrobial efficacy of riboflavin/UVA combination (365 nm) in vitro for bacterial and fungal isolates: a potential new treatment for infectious keratitis. Invest Ophthalmol Vis Sci 2008;49(8):3402-8.

194. Schrier A, Greebel G, Attia H, Trokel S, Smith EF. In vitro antimicrobial efficacy of riboflavin and ultraviolet light on Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, and Pseudomonas aeruginosa. J Refract Surg 2009;25(9):S799-802.

195. Sauer A, Letscher-Bru V, Speeg-Schatz C, Touboul D, Colin J, Candolfi E, Bourcier T. In vitro efficacy of antifungal treatment using riboflavin/UV-A (365 nm) combination and amphotericin B. Invest Ophthalmol Vis Sci 2010;51(8):3950-3.

196. Schnitzler E, Sporl E, Seiler T. [Irradiation of cornea with ultraviolet light and riboflavin administration as a new treatment for erosive corneal processes, preliminary results in four patients]. Klin Monbl Augenheilkd 2000;217(3):190-3.

197. Ehlers N, Hjortdal J, Nielsen K, Sondergaard A. Riboflavin-UVA treatment in the management of edema and nonhealing ulcers of the cornea. J Refract Surg 2009;25(9):S803-6.

198. Micelli Ferrari T, Leozappa M, Lorusso M, Epifani E, Micelli Ferrari L. Escherichia coli keratitis treated with ultraviolet A/riboflavin corneal cross-linking: a case report. Eur J Ophthalmol 2009;19(2):295-7.

199. Moren H, Malmsjo M, Mortensen J, Ohrstrom A. Riboflavin and ultraviolet a collagen crosslinking of the cornea for the treatment of keratitis. Cornea 2010;29(1):102-4.

200. Al-Sabai N, Koppen C, Tassignon MJ. UVA/riboflavin crosslinking as treatment for corneal melting. Bull Soc Belge Ophtalmol 2010(315):13-7.


201. Iseli HP, Thiel MA, Hafezi F, Kampmeier J, Seiler T. Ultraviolet A/riboflavin corneal cross-linking for infectious keratitis associated with corneal melts. Cornea 2008;27(5):590-4.

202. Khan YA, Kashiwabuchi RT, Martins SA, Castro-Combs JM, Kalyani S, Stanley P, Flikier D, Behrens A. Riboflavin and Ultraviolet Light A Therapy as an Adjuvant Treatment for Medically Refractive Acanthamoeba Keratitis Report of 3 Cases. Ophthalmology 2010.

203. Garduno-Vieyra L, Gonzalez-Sanchez CR, Hernandez-Da Mota SE. Ultraviolet-a light and riboflavin therapy for acanthamoeba keratitis: a case report. Case Report Ophthalmol 2011;2(2):291-5.

Publications in the series

Örebro Studies in Medicine

1. Bergemalm, Per-Olof (2004). Audiologic and cognitive long-term sequelae from closed head injury.

2. Jansson, Kjell (2004). Intraperitoneal Microdialysis. Technique and Results.

3. Windahl, Torgny (2004). Clinical aspects of laser treatment of lichen sclerosus and squamous cell carcinoma of the penis.

4. Carlsson, Per-Inge (2004). Hearing impairment and deafness. Genetic and environmental factors – interactions – consequences. A clinical audiological approach.

5. Wågsäter, Dick (2005). CXCL16 and CD137 in Atherosclerosis.

6. Jatta, Ken (2006). Inflammation in Atherosclerosis.

7. Dreifaldt, Ann Charlotte (2006). Epidemiological Aspects on Malignant Diseases in Childhood.

8. Jurstrand, Margaretha (2006). Detection of Chlamydia trachomatis and Mycoplasma genitalium by genetic and serological methods.

9. Norén, Torbjörn (2006). Clostridium difficile, epidemiology and antibiotic resistance.

10. Anderzén Carlsson, Agneta (2007). Children with Cancer – Focusing on their Fear and on how their Fear is Handled.

11. Ocaya, Pauline (2007). Retinoid metabolism and signalling in vascular smooth muscle cells.

12. Nilsson, Andreas (2008). Physical activity assessed by accelerometry in children.

13. Eliasson, Henrik (2008). Tularemia – epidemiological, clinical and diagnostic aspects.

14. Walldén, Jakob (2008). The influence of opioids on gastric function: experimental and clinical studies.

15. Andrén, Ove (2008). Natural history and prognostic factors in localized prostate cancer.

16. Svantesson, Mia (2008). Postpone death? Nurse-physician perspectives and ethics rounds.

17. Björk, Tabita (2008). Measuring Eating Disorder Outcome – Definitions, dropouts and patients’ perspectives.

18. Ahlsson, Anders (2008). Atrial Fibrillation in Cardiac Surgery.

19. Parihar, Vishal Singh (2008). Human Listeriosis – Sources and Routes.

20. Berglund, Carolina (2008). Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus. Epidemiological aspects of MRSA and the dissemination in the community and in hospitals.

21. Nilsagård, Ylva (2008). Walking ability, balance and accidental falls in persons with Multiple Sclerosis.

22. Johansson, Ann-Christin (2008). Psychosocial factors in patients with lumbar disc herniation: Enhancing postoperative outcome by the identification of predictive factors and optimised physiotherapy.

23. Larsson, Matz (2008). Secondary exposure to inhaled tobacco products.

24. Hahn-Strömberg, Victoria (2008). Cell adhesion proteins in different invasive patterns of colon carcinoma: A morphometric and molecular genetic study.

25. Böttiger, Anna (2008). Genetic Variation in the Folate Receptor-α and Methylenetetrahydrofolate Reductase Genes as Determinants of Plasma Homocysteine Concentrations.

26. Andersson, Gunnel (2009). Urinary incontinence. Prevalence, treatment seeking behaviour, experiences and perceptions among persons with and without urinary leakage.

27. Elfström, Peter (2009). Associated disorders in celiac disease.

28. Skårberg, Kurt (2009). Anabolic-androgenic steroid users in treatment: Social background, drug use patterns and criminality.

29. de Man Lapidoth, Joakim (2009). Binge Eating and Obesity Treatment – Prevalence, Measurement and Long-term Outcome.

30. Vumma, Ravi (2009). Functional Characterization of Tyrosine and Tryptophan Transport in Fibroblasts from Healthy Controls, Patients with Schizophrenia and Bipolar Disorder.

31. Jacobsson, Susanne (2009). Characterisation of Neisseria meningitidis from a virulence and immunogenic perspective that includes variations in novel vaccine antigens.

32. Allvin, Renée (2009). Postoperative Recovery. Development of a Multi-Dimensional Questionnaire for Assessment of Recovery.

33. Hagnelius, Nils-Olof (2009). Vascular Mechanisms in Dementia with Special Reference to Folate and Fibrinolysis.

34. Duberg, Ann-Sofi (2009). Hepatitis C virus infection. A nationwide study of assiciated morbidity and mortality.

35. Söderqvist, Fredrik (2009). Health symptoms and potential effects on the blood-brain and blood-cerebrospinal fluid barriers associated with use of wireless telephones.

36. Neander, Kerstin (2009). Indispensable Interaction. Parents’ perspectives on parent–child interaction interventions and beneficial meetings.

37. Ekwall, Eva (2009). Women’s Experiences of Gynecological Cancer and Interaction with the Health Care System through Different Phases of the Disease.

38. Thulin Hedberg, Sara (2009). Antibiotic susceptibility and resistance in Neisseria meningitidis – phenotypic and genotypic characteristics.

39. Hammer, Ann (2010). Forced use on arm function after stroke. Clinically rated and self-reported outcome and measurement during the sub-acute phase.

40. Westman, Anders (2010). Musculoskeletal pain in primary health care: A biopsychosocial perspective for assessment and treatment.

41. Gustafsson, Sanna Aila (2010). The importance of being thin – Perceived expectations from self and others and the effect on self-evaluation in girls with disordered eating.

42. Johansson, Bengt (2010). Long-term outcome research on PDR brachytherapy with focus on breast, base of tongue and lip cancer.

43. Tina, Elisabet (2010). Biological markers in breast cancer and acute leukaemia with focus on drug resistance.

44. Overmeer, Thomas (2010). Implementing psychosocial factors in physical therapy treatment for patients with musculoskeletal pain in primary care.

45. Prenkert, Malin (2010). On mechanisms of drug resistance in acute myloid leukemia.

46. de Leon, Alex (2010). Effects of Anesthesia on Esophageal Sphincters in Obese Patients.

47. Josefson, Anna (2010). Nickel allergy and hand eczema – epidemiological aspects.

48. Almon, Ricardo (2010). Lactase Persistence and Lactase Non-Persistence. Prevalence, influence on body fat, body height, and relation to the metabolic syndrome.

49. Ohlin, Andreas (2010). Aspects on early diagnosis of neonatal sepsis.

50. Oliynyk, Igor (2010). Advances in Pharmacological Treatment of Cystic Fibrosis.

51. Franzén, Karin (2011). Interventions for Urinary Incontinence in Women. Survey and effects on population and patient level.

52. Loiske, Karin (2011). Echocardiographic measurements of the heart. With focus on the right ventricle.

53. Hellmark, Bengt (2011). Genotypic and phenotypic characterisation of Staphylococcus epidermidis isolated from prosthetic joint infections.

54. Eriksson Crommert, Martin (2011). On the role of transversus abdominis in trunk motor control.

55. Ahlstrand, Rebecca (2011). Effects of Anesthesia on Esophageal Sphincters.

56. Holländare, Fredrik (2011). Managing Depression via the Internet – self-report measures, treatment & relapse prevention.

57. Johansson, Jessica (2011). Amino Acid Transport and Receptor Binding Properties in Neuropsychiatric Disorders using the Fibroblast Cell Model.

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