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The variety and complexity of ocular infections have increased significantly in the last decade since the publication of Cumitech 13B, Laboratory Diagnosis of Ocular Infections (L. D. Gray, P. H. Gilligan, and W. C. Fowler, Cumitech 13B, Laboratory Diagnosis of Ocular Infections, 2010). The purpose of this practical guidance document is to review, for individuals working in clinical microbiology laboratories, current tools used in the laboratory diagnosis of ocular infections. This document begins by describing the complex, delicate anatomy of the eye, which often leads to limitations in specimen quantity, requiring a close working bond between laboratorians and ophthalmologists to ensure high-quality diagnostic care. Descriptions are provided of common ocular infections in developed nations and neglected ocular infections seen in developing nations. Subsequently, preanalytic, analytic, and postanalytic aspects of laboratory diagnosis and antimicrobial susceptibility testing are explored in depth.

Human nuclear cataract formation is a multi-factorial disease with contributions to light scattering from many cellular sources that change their scattering properties over decades. The aging process produces aggregation of cytoplasmic crystallin proteins, which alters the protein packing and texture of the cytoplasm. Previous studies of the cytoplasmic texture quantified increases in density fluctuations in protein packing and theoretically predicted the corresponding scattering. Multilamellar bodies (MLBs) are large particles with a core of crystallin cytoplasm that have been suggested to be major sources of scattering in human nuclei. The core has been shown to condense over time such that the refractive index increases compared to the adjacent aged and textured cytoplasm. Electron tomography is used here to visualize the 3D arrangement of protein aggregates in aged and cataractous lens nuclear cytoplasm compared to the dense protein packing in the cores of MLBs. Thin sections, 70 nm thick, were prepared from epoxy-embedded human transparent donor lenses and nuclear cataracts. Tilt series were collected on an FEI T20 transmission electron microscope (TEM) operated at 200 kV using 15 nm gold particles as fiducial markers. Images were aligned and corrected with FEI software and reconstructed with IMOD and other software packages to produce animated tilt series and stereo anaglyphs. The 3D views of protein density showed the relatively uniform packing of proteins in aged transparent lens nuclear cytoplasm and less dense packing of aged cataractous cytoplasm where many low-density regions can be appreciated in the absence of the TEM projection artifacts. In contrast the cores of the MLBs showed a dense packing of protein with minimal density fluctuations. These observations support the conclusion that, during the nuclear cataract formation, alterations in protein packing are extensive and can result in pronounced density fluctuations. Aging causes the MLB cores to become increasingly different in their protein packing from the adjacent cytoplasm. These results support the hypothesis that the MLBs increase their scattering with age and nuclear cataract formation.

Purpose: To characterize multilamellar bodies (MLBs), determine their distribution along the optic axis and predict their potential Mie scattering within human age-related nuclear cataracts. Previous studies restricted to the equatorial plane have shown that MLBs are rare spherical objects that are 1–4 mm in diameter and covered by multiple layers of thin lipid-rich membranes. Methods: Eight human aged transparent lenses were obtained from eye bank donors and eight human age-related nuclear cataracts were obtained immediately after extracapsular extraction. Each sample was Vibratome sectioned fresh into 200 mm thick sections that were fixed and embedded for light or electron microscopy. Light micrograph montages of the optic axis containing the juvenile, fetal and embryonic nuclei were examined. Mie scattering for random coated spherical particles was calculated based on assumed and measured particle parameters. Results: Cells along the optic axis of the cataract contained approximately 7$5 times more MLBs as similar regions of the aged transparent lens, although these MLBs occurred with extremely low frequency. Cells of the aged transparent lens contained 1$3 MLBs mm K2 , while those of the cataract contained 9$6 MLBs mm K2 , which are equivalent to calculated densities of 5$6!10 2 and 4$1!10 3 mm K3 , respectively. While some MLBs were located within the cytoplasm near cell membranes, others were found away from membranes. The MLBs are distinct from circular profiles resulting from finger-like projections between adjacent cells. MLBs displayed varying geometries and cytoplasmic textures, although predominately spherical with interiors similar to adjacent fiber cell cytoplasm. These results are in agreement with previous theoretical analysis of light scattering from human lenses and with previous morphological studies examining the equatorial plane of the lens. Potential Mie scattering of spherical particles with the average properties of the observed MLBs and assumed refractive index properties was calculated to be forward scattering of as much as 20% of the incident light. Conclusions: The observed low frequency and absence of clustering of MLBs in the equatorial plane and along the optic axis suggests that MLBs are most likely uniformly distributed throughout the embryonic, fetal and juvenile nuclei of age-related cataracts. Because of their size, distribution, textured cytoplasm and calculated Mie scattering, MLBs probably cause local fluctuations in refractive index in human lens nuclei and, therefore, are potential sources of low-angle, forward light scattering that could impair image formation. q 2004 Elsevier Ltd. All rights reserved.

Purpose. To evaluate the chicken cornea as a model for corneal wound healing research. Methods. We conducted studies on normal chicken corneas and on corneas following mechanical debridement and photorefractive keratectomy (PRK). Results. The chicken cornea possesses six distinct layers that resemble the layers of the human cornea, including a substantial Bowman’s layer measuring 5.2 ± 0.3µm thick. Reepithelialization time was 44.8 ± 1.1 hours with a sliding rate of 75.3 ± 3.2µm/hour following mechanical debridement and 54.4 ± 2.8 hours with a sliding rate of 63.3 ± 3.2µm/hour for PRK-treated corneas. Biomicroscopic haze post-PRK peaked at 4–6 weeks and regressed until 20 weeks post-PRK. Histologic and clinical observations of wound healing strongly paralleled that noted in humans. Conclusion. The chicken cornea’s morphology and wound healing response render it a useful model for corneal wound healing investigations.

Introduction. Previous studies with the free electron laser have only assessed the corneal tissue response in ex vivo models. This is the first free electron laser investigation that has examined the clinical and histologic response of the cornea in vivo . Methods. The Mark III free electron laser was tuned to 2.94 μm for a total of 212 separate in vivo 1 mm spot ablations at varied fluences (0.4–2.04 J/cm 2 ), repetition rates (1–15 Hz), and number of pulses (1–32). Also, 22 separate in vivo central scanning patterns were performed. Results. For the in vivo chicken cornea, the threshold of ablation is approximately 0.64 J/cm 2 at a repetition rate of 10 Hz. Clinical biomicroscopy and light microscopy revealed parameter-dependent collateral damage (10–40) μm) induced by laser energy. Wound healing following free electron laser ablation exhibits patterns similar to those seen in excimer laser anterior keratectomy, including increased epithelial thickness, new collagen deposition, increased numbers of keratocytes, and patterns congruent with attempted profiles of myopic ablation. Conclusions. With further study, continued parameter adjustment, and refined delivery schemes, the potential for marked improvement and possible application appears promising.

Introduction. In laser vision correction surgery, lasers are generally utilized based on their beam-tissue interactions and corneal absorption characteristics. Therefore, the free electron laser, with its ability to provide broad wavelength tunability, is a unique research tool for investigating wavelengths of possible corneal ablation. Methods. Mark III free electron laser wavelengths between 2.94 and 6.7 μm were delivered in serial 0.1 μm intervals to corneas of freshly enucleated porcine globes. Collateral damage, ablation depth, and ablation diameter were measured in histologic sections. Results. The least collateral damage (12–13 μm) was demonstrated at three wavelengths: 6.0, 6.1 (amide I), and 6.3 μm. Minimal collateral damage (15 μm) was noted at 2.94 μm (OH-stretch) and at 6.2 μm. Slightly greater collateral damage was noted at 6.45 μm (amide II), as well as at the 5.5–5.7 μm range, but this was still substantially less than the collateral damage noted at the other wavelengths tested. Conclusions. Our results suggest that select mid-infrared wavelengths have potential for keratorefractive surgery and warrant additional study. Further, the free electron laser's ability to allow parameter adjustment in the far-ultraviolet spectrum may provide unprecedented insights toward establishing the gold-standard parameters for laser vision correction surgery.

Mycobacterium chelonae is ubiquitous in the environment but is an uncommon cause of ocular and periocular infections. It is a pathogen that has been gaining increased attention in the ophthalmic literature because of the relatively large number of infections associated with laser-assisted in situ keratomileusis and other forms of refractive surgery. The authors present 3 patients who developed canaliculitis culture positive for M. chelonae more than a year after SmartPlug placement. These cases highlight some of the clinical scenarios that may be encountered in those who present with canaliculitis with a history of intracanalicular plug placement. Therapeutic considerations are also suggested.

The purpose is to determine the nature of the cellular rearrangements occurring through the remodeling zone (RZ) in human donor lenses, identified previously by confocal microscopy to be about 100 μm from the capsule. Human donor lenses were fixed with 10% formalin followed by 4% paraformaldehyde prior to processing for transmission electron microscopy. Of 27 fixed lenses, ages 22, 55 and 92 years were examined in detail. Overview electron micrographs confirmed the loss of cellular organization present in the outer cortex (80 μm thick) as the cells transitioned into the RZ. The transition occurred within a few cell layers and fiber cells in the RZ completely lost their classical hexagonal cross-sectional appearance. Cell interfaces became unusually interdigitated and irregular even though the radial cell columns were retained. Gap junctions appeared to be unaffected. After the RZ (40 μm thick), the cells were still irregular but more recognizable as fiber cells with typical interdigitations and the appearance of undulating membranes. Cell thickness was irregular after the RZ with some cells compacted, while others were not, up to the zone of full compaction in the adult nucleus. Similar dramatic cellular changes were observed within the RZ for each lens regardless of age. Because the cytoskeleton controls cell shape, dramatic cellular rearrangements that occur in the RZ most likely are due to alterations in the associations of crystallins to the lens-specific cytoskeletal beaded intermediate filaments. It is also likely that cytoskeletal attachments to membranes are altered to allow undulating membranes to develop.

BACKGROUND: Instrumentation for performing a uniform lamellar keratoplasty has been undergoing various stages of refinement. Reliable reproduction and uniform thickness and diameter of lamellar resections is required before lamellar refractive keratoplasty can be considered safe and effective. METHODS: The authors used the Draeger rotary microkeratome with mechanical blade advance for lamellar dissections in 61 human cadaver eyes prepared by injecting Swinger-Kommehl (SK) solution into the anterior chamber to a pressure of 35 to 40 mm Hg and by soaking for 30 minutes in SK solution. Spacer sizes of 0.25 to 0.40 units were utilized using an anterior lamellar disc diameter estimate between 8.0 and 8.5 mm and a stromal lamellar disc diameter estimate between 5.5 and 6.5 mm. Preoperative pachometry, anterior and stromal lamellar disc thicknesses, and anterior and stromal lamellar disc diameters were measured. RESULTS: The Draeger unit created anterior lamellar thickness between 100 and 268 µm. Stromal lamellar disc thicknesses were consistently between 90 and 161 µG?. The continuous, unidirectional, rotary blade and the uniform mechanical advance of the instrument produced a generally uniform bed as evaluated by scanning electron microscopy, although undulations were still present. CONCLUSION: The Draeger microkeratome produced regular lamellar dissections; however, predictability of the thickness of the lenticules varied 10% to 20%, and of the diameter, 1.5% to 15%. Predictability improved with experience. This variability may reduce predictability of refractive outcome. [Refract Corneal Surg. 1 994; 1 0:49-55.)

PURPOSE To employ Mie scattering theory to predict the light-scattering from micrometer-sized particles surrounded by lipid shells, called multilamellar bodies (MLBs), reported in human age-related nuclear cataracts. METHODS Mie scattering theory is applicable to randomly distributed spherical and globular particles separated by distances much greater than the wavelength of incident light. With an assumed refractive index of 1.40 for nuclear cytoplasm, particle refractive indices from 1.33 to 1.58 were used to calculate scattering efficiencies for particle radii 0.05 to 3 microm and incident light with wavelengths (in vacuo) of 400, 550, and 700 nm. RESULTS Surface plots of scattering efficiency versus particle radius and refractive index were calculated for coated spherical particles. Pronounced peaks and valleys identified combinations of particle parameters that produce high and low scattering efficiencies. Small particles (

An 85-year-old, immunocompetent man was referred to the authors due to the presence of an enlarging, pigmented mass of the conjunctiva concerning for a conjunctival melanoma. Wide excision of the mass revealed a pigmented or "dematiaceous" fungus. He was treated with topical natamycin, and the lesion healed well without any evidence of recurrence. Dematiaceous fungi should be considered in the differential for pigmented conjunctival lesions.

Recent focus on the optical correction for "contact lens failure" keratoconus patients has been in the use of epikeratoplasty as an alternative to penetrating keratoplasty. In a 2-year retrospective review of keratoconus patients, 33 were referred as "contact lens failures" and as such might be considered candidates for surgical intervention. Of these, 29 patients (average keratometry, 50.4; range, 43-73) were successfully refitted with contact lenses. All patients obtained wearing times of 12 hours and achieved a visual acuity of 20/40 or better, with 85% realizing 20/30 or better. The data suggest that with concerted effort most keratoconus patients may be successfully refitted despite initial contact lens failure.