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Objective To investigate the imaging manifestations of different filamentous fungal strains under the confocal laser scanning microscope and slit-lamp microscope,and evaluate the feasibility of rapid diagnosis and therapeutic efficacy judgment for fungal culture negative patients.Methods A diagnosis trail was performed.Nine hundred and ninety-three patients with fungal keratitis (FK) which were varified by fungal culture were included in Henan Eye Hospital from September 2013 to January 2014.Distribution of fungi strains and positive rate of fungal strains by fungal culture and corneal confocal laser scanning microscopy were compared.The imaging characteristics of different filamentous fungi and different stages of one filamentous fungi under the slit-lamp microscope and confocal laser scanning microscopy were summarized.Results In the 993 FK patients,the diagnostic positive rate of fungal culture and confocal laser scanning microscopy was 43.20％ and 82.07％,respectively,showing a significant difference between them (x2 =45.323,P =0.000).In 429 culture-positive patients,the diagnostic positive rate of confocal laser scanning microscopy was 92.31％;while in 564 culture-negative patients,the diagnostic positive rate of confocal laser scanning microscopy was 74.29％.In 429 culture-positive patients,Aspergillus was the most common genus,accounting for 50.12％,and followed by Fusarium sp.and Altemaria sp.(18.18％ and 10.49％).There were no significant differences in fungal species distributions between fungal culture and confocal laser scanning microscopy examination in 429 cases (all at P＞0.05).The imaging characteristics under the slit-lamp microscope and confocal laser scanning microscope were different in different fungi stains.Aspergillus infection showed a plume-like corneal ulcer,and the Aspergillus sp.hyphae were thin and line-shaped with high reflective light and less branched under the confocal laser scanning microscope.Toothpaste-like corneal infiltration was seen in Fusarium sp.-infectious lesions under the slit lamp microscope,and mycelium showed a high-reflective long rod-like image with less branch in the image of confocal laser scanning microscope.Alternaria alternate sp.corneal infection showed nevus lesions,and hyphae characterized by high-reflective long rod or string beads in shape with less branches in the image of confocal laser scanning microscope.The mycelium was ruptured,shorter,thinner with weak reflective light following drug therapy.The differential diagnosis could be easily obtained between hyphae and corneal nerve fibers by confocal laser scanning microscope.Hyphae intertwined,or had branches with diffuse distribution,which surrounded by highreflective inflammatory cells and destructed matrix fiber and were located in stroma.The corneal nerve fibers located between epithelium layer and stroma layer,surrounded by normal epithelium or stroma structure.The diameter of the thicker nerve fibers in the stroma layer was obviously thicker than that of the hyphae.Conclusions The diagnosis rate of confocal laser scanning microscope combined with slit-lamp microscope for filamentous fungi-infectious FK is higher than that of fungal culture.The combination procedure of confocal laser scanning microscope and slit lamp microscope examination provides a rapid evaluation for fungi strains and therapeutic efficacy in the FK patients with negative results by fungal culture.
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Background Xenotransplantation is arousing more attention of researchers because of the lack of corneal donors.Biological corneal scaffolds constructed by porcine corneal acellular stroma appears to have an acceptable biocompatibility.However,its clinical effects and the histomorphological features in the corneal tissue of receipts' are still unclear.Objective This study was to evaluate the viability of bioengineered corneas as a new material of human lamellar keratoplasty and observe the in viva biological features after human keratoplasty under the laser confocal microscope.Methods A prospective serial cases observational study was carried out.Fifteen eyes of 15 patients with infectious keratitis were enrolled in Henan Eye Hospital from February to August 2014 under the approval of Ethic Committee of Henan Eye Hospital and informed consent of each patient, including 4 eyes of fungal keratitis,1 eye of bacterial keratitis, 9 eyes of mixed infectious keratitis and 1 eye of leucoma.Corneal lamellar transplantation was performed on the eyes with the bioengineered corneas as grafts and the follow-up time was one year.The survival of grafts was assessed and scored, and the indices including corneal transparency,neovascularization, graft lysis or inflammatory recurrence and visual acuity (logMAR) were evaluated.The morphology and density of corneal epithelial cells and endothelial cells, corneal stroma and subepithelial neural fibers were examined by laser confocal microscope 3,6,9 and 12 months after surgery.Results Postoperative inflammatory response was seen 3 days and disappeared 7 days after surgery.The grafts were clear 1 month after surgery, and no corneal dissolution was found during the follow-up duration.Glaucoma occurred in 1 eye at 6 months and graft rejection occurred in another eye 12 months after surgery.The logMAR, corneal transparency scores and corneal neovascularization scores improved after surgery in comparison with before surgery,with a significant difference among various time points (x2 =92.63,59.37,10.50 ,all at P＜0.05).Complete epithelization of grafts was seen.Compared with the contralateral eyes,the morphology of epithelial cells was similar and endothelial cells were enlarged in the operated eyes.In addition,no stromal cell structure was seen in the corneal stroma in the operative eyes.Subepithelial nerve fibers appeared in 8 eyes at 6 months, but the fiber density was lower in the operated eyes than that in the contralateral eyes.Significant difference was found in epithelial cell density among different time points (F=1.48, P =0.22).The endothelial cell densities were (2 542 ± 119), (1 895 ± 129), (1 869 ± 135), (1 854 ± 101) and (1 844 ± 103)/mm2 before surgery and 3, 6, 9 and 12 months after surgery, showing significant differences between preoperation and postoperative time points (all at P＜0.05).The subepithelial nerve fibers densities were (1.26± 0.13),(3.62±0.81) and (5.98±0.44) mm/mm2 at 6,9 and 12 months after surgery,with significant differences between adjacent two time points (t'=-8.16 ,-7.24;both at P=0.00).Conclusions Bioengineered corneal grafts survive well in human eye after lamellar transplantation,which can reconstruct ocular surface and improve the visual acuity.Biological cornea can replace human corneal materials.
