Atomic Force Microscopy for the Evaluation of Corneal Surface Roughness after Femtosecond Laser Flap Creation and Excimer Ablation.

Introduction: It is well known that the femtosecond laser lamellar cut has some degree of roughness. Nevertheless, as in femtosecond laser assisted LASIK (FS-LASIK), an excimer LASIK ablation is performed, the post-ablation stromal bed should show a marked decrease in roughness. We decided to compare, using atomic force microscopy (AFM), the roughness of the corneal stromal bed, after a Femtosecond lasers device (FS) flap was created with or without an excimer myopic ablation.Methods: Using 6 freshly enucleated porcine eyes, we created in every eye a flap using a femtosecond laser. Additionally in 3 eyes an excimer laser ablation to correct -3 diopters (D) was made. AFM imaging of the remaining corneal stroma was performed. Ten different square areas of 20μm x 20μm at the central area of the stroma of each corneal sample were studied. The roughness parameters used was the root-mean-square (RMS) deviation from a perfectly flap plane.Results: The RMS deviation were 360 ± 120 nm in femtosecond laser only, and 110 ± 20 nm in those cases where excimer is also involved (p<0.0001).Conclusions: Our results show that the roughness of the surface treated with excimer is clearly lower than in the group with no excimer ablation, thus the application of laser excimer after a flap creation by femtosecond laser flap creation may soften the nano-irregularities created by this technique.

Corneal Culture in Infectious Keratitis: Effect of the Inoculation Method and Media on the Corneal Culture Outcome

Background: To compare two different methods of corneal culture in infectious keratitis: multiple sampling for direct inoculation and enrichment (standard method) and a single sample via transport medium for indirect inoculation (indirect inoculation method). Methods: Prospective inclusion of patients fulfilling predefined criteria of infectious keratitis undergoing corneal culture according to both studied methods in a randomized order. Results: The standard method resulted in a significantly higher proportion of positive culture outcomes among the 94 included episodes of infectious keratitis (61%; 57/94) than the indirect inoculation method (44%; 41/94) (p = 0.002) and a significantly higher proportion of microorganisms than the indirect inoculation method, with a Cohen’s kappa of 0.38 (95% CI: 0.28–0.49) for agreement between the methods. Subanalysis of culture results showed that direct inoculation on gonococcal agar only combined with the indirect inoculation method resulted in a similar rate of culture positive patients and proportion of detected microorganisms to the standard method. Conclusion: Indirect inoculation of one corneal sample cannot replace direct inoculation of multiple corneal samples without loss of information. A combination of directly and indirectly inoculated samples can reduce the number of corneal samples by four without statistically significant differences in culture outcome or in the proportion of detected microorganisms.

FONSECAEA PEDROSOI – A CAUSE OF OCCULAR CHROMOBLASTOMYCOSIS

Fonsecaea pedrosoi (F. pedrosoi) is dematiaceous fungus and is the most common cause for chromoblastomycosis. It affects the exposed skin, mostly of the lower extremities. Arare case of mycotic keratitis was diagnosed in our hospital caused by F. pedrosoi. Corneal sample received in the laboratory was processed by standard mycological methods, F. pedrosoi was isolated, patient was started on antifungals his condition improved and there was no relapse. This case report shows that F. pedrosoi can infect cornea. Further, a prompt diagnosis and vigorous treatment improves patient's clinical condition.

Quantification of double stranded DNA breaks and telomere length as proxies for corneal damage and replicative stress in 64 human keratoconus corneas

PurposeThe pathogenesis of keratoconus (KC) is multifactorial and associated with oxidative stress and subsequent DNA damage. The aim of this study was to investigate differences in DNA damage and replicative stress in patients with KC, and in both healthy and diseased controls.MethodsSixty-four corneal buttons were obtained from 27 patients with KC after corneal transplant surgery, 21 patients with a decompensated graft (DG), and 16 healthy controls (HC). The amount of intact Alu elements per genome copy as measured by qPCR was used to quantify intact DNA. Telomere length was measured as a proxy for replicative stress. In addition, telomerase reverse transcriptase (hTERT) gene expression level was assessed.ResultsMean (±SD) DNA damage was similar between the KC (5.56 ±14.08), DG (3.16 ±8.22), and HC (3.51 ±6.66) groups (P=0.807). No associations were found between DNA damage and patient age (P=0.523), atopic constitution (P=0.240), or contact lens wear (P=0.393). Telomere length differed (P=0.034), most notably in the KC group, and hTERT was not detected in any corneal sample. Three cross-linked (CXL) KC corneas did not contain significant more DNA damage (2.6x, P = 0.750).ConclusionsBased on these findings, differences in actual corneal DNA damage in KC could not be identified, and the longer telomere length in KC did not support replicative stress as a major etiological factor in the pathogenesis of KC. Future longitudinal investigations on KC etiology should assess progressive early cases to better comprehend the cellular and molecular processes preceding the archetypical morphological changes.PrecisOxidative stress is allegedly linked with the development of keratoconus. Whether these stressors actually lead to persisting DNA damage and replicative stress is debated. DNA damage was comparable with control samples, and a shortened telomere length was not identified.

Fertőzéses keratitisek diagnosztikája és kezelése

We summarize up-to-date diagnostic and treatment of infectious keratitis using literature data and some clinical examples. In the clinical practice, most commonly bacterial, herpetic, mycotic and acanthamoeba keratitis occur. Beside slitlamp examination, for diagnostic purpose, we analyse corneal sensitivity, perform in vivo confocal microscopy, polymerase-chain-reaction (PCR), in vitro culture and histological examination of the corneal sample. As conservative treatment we use primarily topical moxifloxacin or cephasolin with fortified tobramycin or gentamycin in bacterial, topical antiviral gel (in some cases in combination with systemic antiviral treatment) in part in combination with topical corticosteroids in herpetic, voriconasole or amphotericin-B in mycotic, and topical-triple-therapy (diamidine, biguanid and antibiotics) in acanthamoeba keratitis. In case of early diagnosis and initiation of topical therapy, most cases of infectious keratitis recover successfully. However, beside conservative treatment, penetrating keratoplasty, amniotic membrane transplantation and crosslinking therapy may be necessary. Crosslinking is solely contraindicated in herpetic keratitis. Orv Hetil. 2017; 158(31): 1203–1212.