A Cluster of Corneal Donor Rim Cultures Positive for Achromobacter Species Associated With Contaminated Eye Solution.

PURPOSE: To investigate a cluster of corneoscleral rim cultures positive for Achromobacter species over a 6-month period at Massachusetts Eye and Ear. METHODS: An increased rate of positive corneal donor rim cultures was noted at Massachusetts Eye and Ear between July and December 2017. Positive cultures were subjected to identification and antimicrobial susceptibility testing by phenotypic (MicroScan WalkAway) and genotypic (16S rDNA sequencing) methods. Samples of the eye wash solution (GeriCare) used in the eye bank were also evaluated. Antimicrobial activity of Optical-GS against Achromobacter spp. at 4°C and 37°C was assessed by time-kill kinetics assay. RESULTS: Of 99 donor rims cultured, 14 (14.1%) grew bacteria with 11 (78.6%) due to uncommon nonfermenting Gram-negative bacilli. These had been identified by standard automated methods as Achromobacter (n = 3), Alcaligenes (n = 3), Ralstonia (n = 2), Pseudomonas (n = 2), and Stenotrophomonas (n = 1). Eight of these 11 isolates were subsequently available for molecular identification, and all were identified as Achromobacter spp. Six bottles of eyewash solution were evaluated and were positive for abundant Achromobacter spp. (3.4 × 105 ± 1.1 CFU/mL). Optisol-GS had no bactericidal activity against Achromobacter spp. at 4°C after 24-hour incubation but was bactericidal at 37°C. None of the patients who had received the contaminated corneas developed postoperative infection. CONCLUSIONS: An eyewash solution arising from a single lot was implicated in the contamination of donor rims by Achromobacter spp. The isolates were able to survive in the Optisol-GS medium at the recommended storage temperature. This highlights the need to continue improving protocols for tissue preparation and storage.

Identification and Visualization of a Distinct Microbiome in Ocular Surface Conjunctival Tissue.

Purpose: Knowledge of whether microorganisms reside in protected niches of the conjunctiva is potentially significant in terms of minimizing risks of contact lens inflammation/infection and endophthalmitis. We define if and how microbial communities from limbal and forniceal conjunctival tissue differ from those on the conjunctival surface. Methods: Human limbal and forniceal conjunctival tissue was obtained from 23 patients undergoing pterygium surgery and analyzed with data from a recent study of conjunctival surface swabs (n = 45). Microbial communities were analyzed by extracting total DNA from tissue samples and surface swabs and sequencing the 16S rRNA gene using the Illumina MiSeq platform. Sequences were quality filtered, clustered into operational taxonomic units (OTUs) at 97% similarity. OTUs associated with blank extraction and sampling negative controls were removed before analysis. Fluorescent in situ hybridization (FISH) was performed on cyrosections of limbal and forniceal conjunctival tissue. Results: There was a significant difference in bacterial community structure between the conjunctival surface and fornix (P = 0.001) and limbus (P = 0.001) tissue. No difference was found in bacterial communities between the limbus and fornix (P = 0.764). Fornix and limbal samples were dominated by OTUs classified to the genus Pseudomonas (relative abundance 79.9%), which were found only in low relative abundances on conjunctival surfaces (6.3%). Application of FISH showed the presence of Pseudomonas in the forniceal tissue sample. Conclusions: There is a discrete tissue-associated microbiome in freshly-collected human limbal and fornix tissue, which is different from the microbial community structure and composition of the ocular surface microbiome.

A case of keratitis associated with limbal relaxing incision.

We report a case of keratitis associated with limbal relaxing incision (LRI). The patient presented with progressive loss of vision with best-corrected visual acuity 20/40. Immature cataract with 1.43D against the rule astigmatism was noted. Prophylactic topical antibiotic was administered before surgery. He underwent uneventful phacoemulsification with intraocular lens implantation with LRI. On the 33rd postoperative day (POD), he presented with infiltrate along LRI site with mild iritis. Corneal scraping was positive for Staphylococcus aureus. After the treatment with topical moxifloxacin and fortified cefazolin, the infiltrate started to resolve. On the 50th POD, the corneal infection was resolved with marked thinning at LRI site.

Regeneración de la superficie ocular: stem cells/células madre y técnicas reconstructivas / Regeneration of the ocular surface: stem cells and reconstructive techniques

La córnea es un tejido transparente constituido microscópicamente por 5 capas bien diferenciadas. El epitelio corneal es esencial para la transparencia corneal y se encuentra en continua renovación a lo largo de la vida a partir de la población de células madre limbocorneales. La localización de estas células madre limbocorneales parece residir en las capas basales del epitelio limbocorneal, de vital importancia para mantener el microambiente de estas células madre limbocorneales, que depende de una variedad de factores intrínsecos y extrínsecos. La insuficiencia límbica se produce cuando ocurre una pérdida parcial o total de estas células madre limbocorneales. Este cuadro lleva a una opacificación corneal con la consiguiente pérdida de visión. En estos casos, el trasplante corneal supone únicamente un reemplazo temporal del epitelio corneal; es necesario llevar a cabo un tratamiento previo con trasplante de limbo autólogo o alogénico, que permita regenerar la población de células limbocorneales dañadas. Para disminuir el riesgo que supone el trasplante de limbo en el ojo donante, se han propuesto técnicas de cultivo de células limbocorneales a partir de pequeñas biopsias limbocorneales (AU)

The cornea is a transparent tissue microscopically constituted by 5 well differentiated layers. The corneal epithelium is essential for corneal transparency and is found in a state of constant renovation throughout life on the basis of the population of limbocorneal stem cells. The localisation of these limbocorneal stem cells seems to be in the basal layers of the limbocorneal epithelium, of vital importance for maintaining the micro-environment of these limbocorneal stem cells, which depend on a variety of intrinsic and extrinsic factors. Limbic insufficiency occurs when there is a partial or total loss of these limbocorneal stem cells. These clinical features lead to a corneal clouding with a resulting loss of vision. In these cases, corneal transplant only represents a temporary replacement of the corneal epithelium; it is necessary to carry out a prior treatment involving transplant of the autologous or allogeneic limbus, which enables regeneration of the population of damaged limbocorneal cells. To reduce the risk involved in the transplant of the limbus of the donor eye, techniques of cultivation of limbocorneal cells on the basis of small limbocorneal biopsies are proposed (AU)

Functions of MUC16 in corneal epithelial cells.

PURPOSE: The membrane-associated mucin MUC16, a heavily O-glycosylated transmembrane protein, is expressed by the ocular surface epithelia and localized on the tips of the surface microplicae. Although its functions in the ocular surface glycocalyx are unknown, it is thought that MUC16 provides a disadhesive barrier to the epithelial membrane. Two other membrane-associated mucins expressed by ocular surface epithelia, MUC1 and MUC4, are multifunctional and have signaling capabilities through their cytoplasmic tails and EGF-like domains, respectively. The MUC16 cytoplasmic tail has not been characterized, but, because it contains a polybasic amino acid sequence, it potentially interacts with the actin cytoskeleton through ezrin/radixin/moesin (ERM) actin-binding proteins. METHODS: The interaction of MUC16 with the actin cytoskeleton through ERMs was investigated using cytoplasmic tail peptides and ERM pull-down experiments. MUC16 functions were determined using RNA interference in immortalized human corneal-limbal epithelial (HCLE) cells. The effect of MUC16 knockdown on microplicae structure in HCLE cells was determined using scanning and immunoelectron microscopy. HCLE cells were incubated with rose bengal dye to measure the role of MUC16 in ocular surface barrier function. Binding of fluorescently labeled Staphylococcus aureus to HCLE cells was measured to determine the role of MUC16 in the protection of pathogen adherence on the ocular surface epithelium. RESULTS: MUC16 cytoplasmic tail peptides bound the N-terminus of ERMs, with no detectable binding of MUC1 and MUC4 peptides. No effect on surface membrane projections could be detected in HCLE cells after MUC16 suppression; however, HCLE cells incubated with rose bengal showed that exclusion of the dye was significantly reduced in cells with MUC16 suppression. In addition, S. aureus binding to HCLE cells was significantly increased with MUC16 suppression. CONCLUSIONS: These results suggest that MUC16 is a multifunctional molecule linked to the actin cytoskeleton. The expression of MUC16 in the ocular surface glycocalyx helps provide a disadhesive protective barrier for the epithelial surface.

Limbal and choroidal Cryptococcus infection in the acquired immunodeficiency syndrome.

PURPOSE: A 30-year-old patient with the acquired immunodeficiency syndrome (AIDS) had limbal nodules and multifocal choroidal lesions. METHODS: A biopsy of the limbal nodules was performed. RESULTS: The biopsy showed Cryptococcus neoformans surrounded by thick mucinous capsules without inflammatory cell infiltration. CONCLUSION: In the differential diagnosis of limbal mass in patients with AIDS, cryptococcal infection should be considered.

Histological and immunopathological analysis of T-cells mediating murine HSV-1 keratitis.

BACKGROUND: Thymus-derived lymphocytes play a critical role in the development of herpes simplex keratitis (HSK). T-cell subsets defined by their expression of various T-cell receptor (TCR) V beta segments were studied following corneal HSV-1 infection (p.i.). METHODS: Conjunctiva, corneal limbus and corneal stroma of two inbred BALB/c congenic mouse strains which differ only in the gene products closely linked to the Igh-1 locus on chromosome 12 were analyzed. RESULTS: While C.B-17 mice (Igh-1b) were resistant to HSK, C.AL-20 mice (Igh-1d) clinically developed severe necrotizing keratitis by day 11 p.i. The corneal stroma of C.B-17 mice remained clear, while it was increasingly infiltrated by mononuclear cells and neutrophils in C.AL-20 mice by day 11 p.i. In C.B-17 mice, Thy1.2+ cells were found in the conjunctiva between days 2 to 4 p.i., and subsequently decreased. Only a few Thy1.2+ cells were found in the limbus, and no such cells were found in the stroma. In contrast, in C.AL-20 mice the numbers of Thy1.2+ cells (activated CD4+, V beta 8+ T cells) profoundly increased in the conjunctiva by day 4 p.i. These cells infiltrated the limbus between days 7 and 11 p.i. and eventually entered the stromal tissue by day 11 p.i. CONCLUSIONS: Our data suggest that the HSV-1-induced corneal tissue destruction is mediated by mononuclear cells and neutrophils and that these cells are probably attracted into the cornea by cytokines elaborated by activated CD4+, V beta 8+ T cells.

Effects of povidone-iodine chemical preparation and saline irrigation on the perilimbal flora.

PURPOSE: To analyze the effects of 5% povidone-iodine preparation and saline irrigation on the species composition of perilimbal flora. METHODS: Cultures were taken from the perilimbal conjunctiva in 100 eyes before preparation for ophthalmic surgery, after instillation of povidone-iodine solution, and after saline irrigation. RESULTS: Bacteria were isolated in 75% of eyes before preparation, in 28% after povidone-iodine instillation, and in 24% after saline irrigation. Fifty-one culture-positive eyes became negative with povidone-iodine, while only four culture-negative eyes became culture-positive (P < 0.001). The number of eyes yielding coagulase-negative staphylococci, Staphylococcus aureus, and Propionibacterium were significantly decreased after povidone-iodine instillation. Twenty-three culture-positive eyes became negative after saline irrigation, while 19 culture-negative eyes became culture-positive (P > 0.25). CONCLUSION: Povidone-iodine solution is effective in reducing bacterial recovery from the perilimbal conjunctiva, where most incisions for intraocular surgery occur. Saline irrigation after povidone-iodine preparation has no significant effect.

Epibulbar molluscum contagiosum in acquired immune deficiency syndrome. Case report and review of the literature.

BACKGROUND: While molluscum contagiosum of the eyelid skin is commonly complicated by conjunctivitis, primary involvement of the conjunctiva or cornea by molluscum lesions is exceedingly rare. The authors studied a 34-year-old man with acquired immune deficiency syndrome (AIDS) in whom multiple molluscum lesions of the lids and a single nodule of the limbus developed. METHODS: The nodular lesion was excised from the limbus and processed for histologic examination by light microscopy. Pertinent literature concerning epibulbar molluscum contagiosum was reviewed. RESULTS: Histopathology of the excised lesion showed molluscum bodies within the acanthotic, hyperkeratotic conjunctival epithelium with surrounding chronic, nongranulomatous inflammation. Only 10 previous cases of conjunctival or corneal primary lesions have been reported, half of which showed associated cutaneous involvement. Lesions tended to be single, noninflamed, dome-shaped, and umbilicated, often with a yellowish central core. Patients were otherwise well and ranged in age from 3 to 55 years. Simple excision was effective in eradicating the lesions. CONCLUSION: Primary epibulbar molluscum contagiosum is rare. Although cutaneous molluscum is common in AIDS, this report is the first to document conjunctival molluscum in a patient with AIDS.