Smurf2 exerts neuroprotective effects on cerebral ischemic injury.

The present study aimed to explore specific mechanisms involved in mediating the neuroprotective effects of Smad ubiquitination regulatory factor 2 (Smurf2) in cerebral ischemic injury. A middle cerebral artery occlusion (MCAO) mouse model and an oxygen-glucose deprivation (OGD)-treated neuron model were developed. The expression of Smurf2, Yin Yang 1 (YY1), hypoxia-inducible factor-1 alpha (HIF1α), and DNA damage-inducible transcript 4 gene (DDIT4) was analyzed. Thereafter, the expression of Smurf2, YY1, HIF1α, and DDIT4 was altered in the MCAO mice and OGD-treated neurons. Apoptosis in tissues and cerebral infarction were assessed. In neurons, the expression of apoptosis-related proteins, viability, and apoptosis were assessed, followed by evaluation of lactate dehydrogenase leakage rate. The interaction between Smurf2 and YY1 was analyzed by coimmunoprecipitation assay and that between YY1 ubiquitination by in vivo ubiquitination experiment. The results showed downregulation of Smurf2 and upregulation of YY1, HIF1α, and DDIT4 in both MCAO mice and OGD-treated neurons. Smurf2 elevated YY1 ubiquitination and degradation, and YY1 increased HIF1α expression to promote DDIT4 in neurons. Overexpressed Smurf2 or downregulated YY1, HIF1α, or DDIT4 reduced the volume of cerebral infarction and apoptosis in MCAO mice, while enhancing cell viability and reducing apoptosis and lactate dehydrogenase leakage in OGD-treated neurons. In summary, our findings elucidated a neuroprotective role of Smurf2 in cerebral ischemic injury via inactivation of the YY1/HIF1α/DDIT4 axis.

Microbial spectrum and antibiotic sensitivity in infantile dacryocystitis.

PURPOSE: To identify the characteristics and the spectrum of microbial agents of infantile dacryocystitis and to assess the trends in both antibiotic sensitivities and pathogens over the past 10 years. METHODS: The microbial and medical records of 546 culture-proven patients (546 eyes) of infantile dacryocystitis diagnosed at Henan Eye Hospital between January 2009 and December 2018 were retrospectively reviewed. Patient demographics, microbial analysis, and susceptibility rates to various antibiotics were done. A chi-squared test for trends was applied to evaluate changes in antibiotic susceptibility and microbial spectrum over time. RESULTS: A total of 546 patients with infantile dacryocystitis were documented. The average age was 2.97 ± 4.15 months, and 42.7% were female. The proportion of gram-positive microbes, gram-negative microbes, and fungi was 80.2, 19.4, and 0.4%, respectively. Minocycline was sensitive to gram-positive bacteria (98.0%). Imipenem was sensitive to gram-negative bacteria (89.2%). Increasing susceptibility was observed in two bacterial isolates: Staphylococcus aureus (P = 0.005) and Streptococcus mitis (P = 0.001). Decreasing susceptibility was observed in one bacterial isolate: Staphylococcus epidermidis (P < 0.0001). Increasing microbial susceptibility over time was detected for 12 antibiotics. Decreasing microbial sensitivity was observed for one antibiotic. CONCLUSIONS: The most common cause of infantile dacryocystitis is Staphylococcus epidermidis. Though a significant trend towards increasing microbial sensitivity to some antibiotics was observed, including glycopeptides, cephalosporins, fluoroquinolones, tetracyclines, and lincosamides, a significant trend towards decreasing microbial sensitivity to amikacin was also detected.

CADM1 enhances intestinal barrier function in a rat model of mild inflammatory bowel disease by inhibiting the STAT3 signaling pathway.

Cell adhesion molecule 1 (CADM1) is frequently silenced in lung, prostate, liver, stomach, pancreatic and breast carcinomas and other forms of human carcinomas. However, it is unclear regarding the role of CADM1 in irritable bowel syndrome with diarrhoea (IBS-D) that is the most common gastrointestinal diagnosis and may contribute to impaired intestinal barrier function. The aim of the present study is to explore the potential mechanism of CADM1 in regulating intestinal barrier function in IBS-D. A rat model with IBS-D induced by the combination method of mother-infant separation, acetic acid and restraint stress was initially established. The defecation frequency, faecal water content (FWC), total intestinal permeability, sIgA, endotoxin, D-lactic acid and diamine oxidase (DAO) were then measured. Next, positive expression of CADM1 protein was detected in distal colonic tissue of rats by immunohistochemistry. The expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in distal colonic mucosa, CADM1, Janus kinase 1 (JAK1), STAT3, p-JAK1, p-STAT3, Claudin-1and Claudin-2 were evaluated using ELISA, RT-qPCR and western blot analysis. IBS-D rats exhibited low CADM1 expression and activated STAT3 signaling pathway. Overexpression of CADM1 in rats was shown to increase Claudin-1 expression, while decreasing expression of STAT3, Claudin-2, TNF-α and IL-6. In addition, silencing of CADM1 or inhibition of the STAT3 signaling pathway was demonstrated to improve the intestinal barrier function. Our study provides evidence that CADM1 can potentially improve intestinal barrier function in rats with IBS-D by inhibiting the STAT3 signaling pathway.

In Vitro Antimicrobial Activity of Diacerein on 76 Isolates of Gram-Positive Cocci from Bacterial Keratitis Patients and In Vivo Study of Diacerein Eye Drops on Staphylococcus aureus Keratitis in Mice.

Bacterial keratitis is an aggressive infectious corneal disease. With the continuing rise in antibiotic resistance and a decline in the discovery of new antibiotics, new antimicrobial drugs are now required. In the present study, we determined the antibacterial activity of diacerein, an anti-inflammatory drug, against 76 Gram-positive cocci isolated from bacterial keratitis patients in vitro and anti-Staphylococcus aureus activity in a mouse bacterial keratitis model in vivo The MICs of diacerein were tested using the broth microdilution method in vitro A BALB/c Staphylococcus aureus keratitis animal model was selected and the corneal clinical observation, viable bacteria, and hematoxylin-eosin and Gram staining of infected corneas were measured to evaluate the antibacterial efficacy of diacerein eye drops in vivo An in vivo eye irritation study was carried out by a modified Draize test in rabbits. Our in vitro results showed that diacerein possesses satisfactory antibacterial activity against the majority of Gram-positive cocci (60/76), including all 57 tested Staphylococcus spp. and 3 Enterococcus spp. The in vivo experiment showed that diacerein eye drops reduced bacterial load and improved ocular clinical scores after topical administration of diacerein drops on infected corneas. The ocular irritation test revealed that diacerein eye drop had excellent ocular tolerance. These results indicated that diacerein possesses in vivo anti-Staphylococcus aureus activity. We suggest that diacerein is a possible topically administered drug for Staphylococcus aureus-infected patients, especially those with ocular surface inflammatory disorders.

γδ T cells regulate the expression of cytokines but not the manifestation of fungal keratitis.

As an important immunoregulatory cell type, the role of γδ T cells in fungal keratitis (FK) is unclear. We observed the distribution of γδ T cells in infected corneas in vivo by two-photon microscopy. The γδ T cells were depleted by neutralizing antibodies. The cytokine expression profile was obtained by protein arrays to determine the cytokines regulated by γδ T cells. ICAM-1, MIP-2 and IL-17A were evaluated by ELISA assays to confirm the role of γδ T cells in FK. We counted the number of neutrophils, evaluated the volume of fungal hyphae and analyzed the manifestation of the disease. The γδ T cells increased significantly at 36 h and 72 h post fungal infection (P < 0.05) and migrated from the limbus to the infection site. The neutralizing antibodies completely depleted the γδ T cells in 24 h. The depletion of γδ T cells led to up regulation of 25 cytokines and down regulation of 3 cytokines. ICAM-1, MIP-2 and IL-17A changed significantly because of the depletion of γδ T cells (P < 0.05). However, the number of neutrophils, volume of fungal hyphae and manifestation of the disease was not affected by the depletion of γδ T cells. Our results demonstrated that γδ T cells have a role in FK via regulation of some cytokines but did not affect the manifestation of this disease, suggesting that γδ T cells are not the key regulator cells in this disease.

[Molecular identification and in vitro susceptibility of Fusarium from fungal keratitis in central China].

OBJECTIVE: To detect the genotypes and in vitro antifungal susceptibility of Fusarium isolated from patients with fungal keratitis in central China. METHODS: Partial translation elongation factor (EF) 1-α of 758 strains of Fusarium isolated from patients with fungal keratitis in Henan Eye Institute during 2002 to 2011 were sequenced. Species and genotypes of Fusarium were identified by conducting BLAST searches of the Fusarium ID database with partial EF1-α sequences as the query. The minimum inhibitory concentrations (MIC) of vorionazole, ketoconazole, terbinafine, natamycin, 5-flucytosine, fluconazol, amphotericin B, nystatin, econazole, clotrimazole, miconazole and itraconazole to 145 isolates of Fusarium were determined by microbroth dilution method according to Clinical Laboratory Standards Institute (CLSI) M38-A program. RESULTS: Among the 758 strains of Fusarium isolates, species of 653 strains were identified. 99.69% of the Fusarium strains were identified by EF1-asequences as Fusarium solani species complex (FSSC), Fusarium oxysporum species complex (FOSC) and Gibberella fujikuroi species complex (GFSC), 0.31% as Fusarium sp. Among the 653 isolates from cornea, FSSC was the predominant Fusarium, 386 isolates (59.11%), with 43 genotypes. The most common seen FSSC genotype was FSSC5-d (132/20. 21%), followed by FSSC3+4-eee (58/8.88%), FSSC3+4-ii (37/5.67%) and FSSC3+4-z (31/4.75%). The second complex was GFSC, 254 isolates (38.90%), with 3 species which were F.proliferatum (124 strains/18.99%), F.verticillioides (112 strains/17.15%) and GFSC (18 strains/2.76%) respectively. The third complex was FOSC, 11 (1.68%) strains, with 6 genotypes. The results of in vitro drug sensitivity test showed that Fusarium strains were sensitive to natamycin, vorionazole and amphotericin B, resistant to 5-fluorocytosine, fluconazole, nystatin, clotrimazole, miconazole. More than 50% of Fusarium strains were sensitive to econazole, ketoconazole, itraconazole and terbinafine. The MIC50 of FSSC to vorionazole, miconazole, terbinafine, Econazole and natamycin was higher than that of F.verticillioides, F.proliferatum and GFSC respectively. CONCLUSIONS: The predominant Fusarium complex of fungal keratitis in central China was FSSC, followed by GFSC. 43 genotypes were included in FSSC in which the most common seen FSSC genotype was FSSC5-d, followed by FSSC3+4-eee, FSSC3+4-ii and FSSC3+4-z. GFSC contained 3 species which were F.proliferatum, F.verticillioides and GFSC respectively. Different genotypes of Fusarium from keratitis had different susceptibility to vorionazole, terbinafine, natamycin and miconazole.

Effects of contact lens solution disinfectants against filamentous fungi.

PURPOSE: To assess and compare the antifungal activity of polyhexamethylene biguanide (PHMB), thimerosal, cetylpyridinium chloride, and chlorhexidine, which are disinfectants used in multipurpose disinfectant solutions (MPDSs) against ocular pathogenic Fusarium solani and Aspergillus flavus isolates in vitro. METHODS: The in vitro activity of PHMB, thimerosal, cetylpyridinium chloride, and chlorhexidine was assessed against 40 isolates of ocular pathogenic fungi that included 24 F. solani and 16 A. flavus isolates. The strains were tested by broth dilution antifungal susceptibility testing of filamentous fungi approved by the CLSI (Clinical and Laboratory Standards Institute) M38-A document. RESULTS: MIC90 (minimum inhibitory concentration for 90% of the organisms) values of PHMB were 4 and 16 µg/mL for F. solani and A. flavus, respectively. MIC90 values of thimerosal were 0.0313 and 0.0625 µg/mL for F. solani and A. flavus, respectively. MIC90 values of cetylpyridinium chloride were 2 and 2 µg/mL for F. solani and A. flavus, respectively. MIC90 values of chlorhexidine were 32 and 32 µg/mL for F. solani and A. flavus, respectively. CONCLUSIONS: As a disinfectant used in MPDSs, thimerosal showed the highest levels of antimicrobial activity against ocular pathogenic F. solani and A. flavus isolates. The concentrations of PHMB (0.0001%), cetylpyridinium chloride (0.00014%), and chlorhexidine (0.003%) in MPDSs are sublethal levels for ocular pathogenic F. solani and A. flavus isolates. Although multiple ingredients within MPDSs play a role in antimicrobial efficacy, antimicrobial activity may be significantly influenced by the disinfectants used in the solution formulations.

The measurement of corneal thickness from center to limbus in vivo in C57BL/6 and BALB/c mice using two-photon imaging.

The mouse corneal thickness is very important for research into the fields of eye disease. However, the in vivo corneal thickness for the entire cornea from the pupil to the limbus was not determined. We measured in vivo corneal layer thicknesses in different corneal areas, from the central cornea to the limbus, in the widely used inbred C57BL/6 and BALB/c mouse strains using two-photon (2 PH) imaging. Eight corneas of the C57BL/6 or BALB/c were scanned using a 2 PH laser scanning fluorescence microscopy system. A total of 14 thicknesses of the different corneal layers, from different corneal regions, were measured using image processing software. In both C57BL/6 and BALB/c mice, the thickness of the corneal layers was inhomogeneous in different areas of the cornea, and all of the layers had their minimum thickness at the limbus. In C57BL/6 mice, the thickness of the corneal layers gradually increased from the central to the paracentral cornea, peaked at the fifth measurement point in the paracentral area, and decreased from this point to the limbus. In BALB/c mice, the thickness of the entire cornea and corneal epithelium had its maximum at the central cornea and gradually decreased from the central cornea to the peripheral cornea and to the limbus. The thickness of the corneal stroma and endothelium had its maximum at the fourth measurement point in the paracentral cornea and gradually decreased from the paracentral cornea to the limbus. The ratio of epithelial thickness to the total corneal thickness gradually decreased from the central cornea to the limbus in both C57BL/6 and BALB/c mice. The minimum ratio was observed at the fourteenth measurement point in both C57BL/6 and BALB/c mice. The ratio of stromal and endothelial to the total corneal thickness gradually increased from the central cornea to the limbus in both C57BL/6 and BALB/c mice. The maximum ratio was observed at the fourteenth measurement point in C57BL/6 mice. The ratio at the first eight measurement points was significantly lower in BALB/c than in C57BL/6 mice (P < 0.05). Our study demonstrated that the thickness of the entire cornea, the corneal epithelium, the corneal stroma and the endothelium was inhomogeneous in different areas of the cornea. Moreover, all of the layers exhibited a minimum thickness at the limbus in both C57BL/6 and BALB/c mice. Furthermore, the corneal thickness in different areas varied between C57BL/6 and BALB/c mice, and the variation in thickness with respect to corneal location for these strains was dissimilar. When using the mouse as an animal model to examine the cornea, it is important to note the differences between humans and mice.

In vitro activity of phenylmercuric acetate against ocular pathogenic fungi.

OBJECTIVES: To determine the antifungal activity of phenylmercuric acetate against ocular pathogenic fungi in vitro and develop new antifungal eye drops to combat keratomycosis. METHODS: The in vitro activity of phenylmercuric acetate was assessed against 261 isolates of ocular pathogenic fungi that included 136 Fusarium spp. isolates, 98 Aspergillus spp. isolates, 10 Alternaria alternata isolates and 17 other pathogens. The activity of phenylmercuric acetate was compared with the activities of amphotericin B and natamycin. In vitro susceptibility testing was performed by broth microdilution assay, in accordance with the CLSI (formerly NCCLS) M38-A guidelines for filamentous fungi. RESULTS: MIC90s of phenylmercuric acetate were 0.0156, 0.0156, 0.0156 and 0.0156 mg/L for Fusarium spp., Aspergillus spp., A. alternata and other pathogens, respectively. MIC90s of amphotericin B were 2, 2, 1 and 1 mg/L for Fusarium spp., Aspergillus spp., A. alternata and other pathogens, respectively. MIC90s of natamycin were 8, 32, 4 and 4 mg/L for Fusarium spp., Aspergillus spp., A. alternata and other pathogens, respectively. CONCLUSIONS: Phenylmercuric acetate has promising antifungal activity, which is significantly superior to the activities of amphotericin B and natamycin against a wide variety of ocular pathogenic fungi based on comparative MIC values. Additional evaluation is required to determine its clinical utility.

[The changes of ocular bacterial isolates and in vitro antimicrobial susceptibility in the past six years].

OBJECTIVE: To analyze the changes of ocular bacterial isolates and their susceptibility to ciprofloxacin, ofloxacin, levofloxacin, gatifloxacin and tobramycin in Henan Province in the past six years. METHODS: Retrospective study of ocular bacterial isolates and their in vitro antimicrobial susceptibility test results of Henan Eye Institute in the past six years. RESULTS: A total of 2044 bacterial isolates were classified into 39 kinds in the past six years, which were mainly from the conjunctival sac. Staphylococcus epidermidis was the most common, followed by Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus cereus. All kinds of the ocular bacteria had the highest susceptibility to gatifloxacin (92.3%) among the tested drugs. Rods were more sensitive to quinolone drugs than cocci and gram-positive rods were more sensitive to tobramycin than the other types of germs. The drug's susceptibility of the four kinds of quinolone except ciprofloxacin decreased year by year with a decreasing ladder fashion every two years in the susceptibility change of levofloxacin in the past six years, while the susceptibility of tobramycin increased slowly from 2004 to 2008 and then decreased. Staphylococcus epidermidis susceptibility to every drug had the similar trend with the general changes in the past six years, that is, their susceptibility to ofloxacin decreased significantly since 2008, their susceptibility to gatifloxacin and tobramycin reduced significantly in 2009. Although Pseudomonas aeruginosa susceptibility to all the drugs had no significant change, for all gram-negative rods, their susceptibility to the four kinds of quinolone drugs were higher than their susceptibility to tobramycin while their susceptibility to levofloxacin and gatifloxacin reduced significantly as time passed by, especially from the year 2008 to 2009. CONCLUSIONS: The most common ocular bacterial isolates were Staphylococcus epidermidis, followed by Pseudomonas aeruginosa. Ocular bacterial isolates' susceptibility to gatifloxacin in vitro was significantly higher than to other drugs in every year but decreased significantly in 2009 while their susceptibility to ofloxacin and levofloxacin decreased significantly since 2008. Their susceptibility to tobramycin increased slowly from 2004 to 2008 and then decreased.

[The investigation of microbial culturing of microkeratome blades and sponges used in laser in situ keratomileusis].

OBJECTIVE: The microbial culturing results were analysed from samples of the microkeratome blade and sponges in laser in situ keratomileusis (LASIK) procedures as for crossing the rational antibiotic eye drops for preventing infectious keratitis. METHOD: In this prospective study, 106 microkeratome blades and 212 sponges were cultured in routine LASIK procedure, at Excimer laser center in Henan Eye Institute During March to April 2009. Positive cultures were then sent for routine sensitivities and the results were analysed. RESULTS: 8 of the 106 blades were culturing positive, the positive rate was 7.55%. Each four positive cultures were in male and female patients. There was no statistical difference with gender (P = 1.000). 23 of the 212 sponges cultures were positive. The positive rate was 10.38%. All positive cultures grew Staphylococcus epidermidis. Twelve sponge positive cultures were in right eye and 11 were in left eye. There was no statistical difference between the right eye and left eye (P = 0.825). All of the 31 positive cultures were sensitive to gatifloxacin. The sensitivity of gatifloxacin, tobramycin, levofloxacin, ofloxacin, and ciprofloxacin were 100.00%, 96.77% (30/31), 93.55% (29/31), 90.32% (28/31) and 74.20% (23/31) respectively. All the patients have no infectious keratitis followed-up more then 6 monthes. CONCLUSION: There could be positive cultures from samples of the microkeratome blade and sponges in routine LASIK procedures but no patients with positive cultures developed postoperative infectious keratitis. The main positive bacteria was Staphylococcus epidermidis. They are sensitive to third and the fourth-generation fluoroquinolones and tobramycin antibiotics. Pre and post-operative supply of sensitive antibiotics can prevent post-operative infection.

A Case Report of Gemella haemolysans Keratitis After Refractive Surgery.

PURPOSE: Systematic review of Gemella haemolysans infection associated with ophthalmology, and to summarize the clinical characteristics of Gemella haemolysan s keratitis after refractive surgery. METHODS: Case report and literature review. RESULTS: We report an 18-year-old man who developed corneal infection after Trans-PRK, and the culture results of lesion specimens confirmed G. haemolysans keratitis. He was treated with fortified topical antibiotics, and clinical improvement was noted shortly after treatment. Resolution of keratitis was achieved at 1 month. Then, a systematic review of the reported cases of ocular G. haemolysans infection was conducted. We summarized clinical manifestations of G. haemolysans infection in cornea. CONCLUSION: We reported a case of G. haemolysans keratitis infection after refractive surgery, and reviewed the literature of ocular G. haemolysans infection.

In vitro activity of thimerosal against ocular pathogenic fungi.

The in vitro activity of thimerosal versus those of amphotericin B and natamycin was assessed against 244 ocular fungal isolates. The activity of thimerosal against Fusarium spp., Aspergillus spp., and Alternaria alternata was 256 times, 512 times, and 128 times, respectively, greater than that of natamycin and 64 times, 32 times, and 32 times, respectively, greater than that of amphotericin B. Thimerosal's antifungal activity was significantly superior to those of amphotericin B and natamycin against ocular pathogenic fungi in vitro.

[Activity of butenafine against ocular pathogenic filamentous fungi in vitro].

OBJECTIVE: To investigate antifungal activity of butenafine in comparison with that of natamycin, amphotericin B and fluconazole against ocular pathogenic filamentous fungi in vitro. METHODS: It was an experimental study. Susceptibility tests were performed against 260 isolates of ocular pathogenic filamentous fungi by broth dilution antifungal susceptibility test of filamentous fungi approved by the Clinical and Laboratory Standards Institute (CLSI) M38-A document. The isolates included Fusarium spp. (136), Aspergillus spp. (98), Alternaria alternata (9), Curvularia lunata (3), and unusual ocular pathogens (14). Final concentration ranged from 0.008 to 16.000 mg/L for butenafine, from 0.031 to 16.000 mg/L for amphotericin B and natamycin, and from 0.5 to 256.0 mg/L for fluconazole. Following incubation at 35 degrees C for 48 h, minimal inhibitory concentration (MIC) was determined according to the CLSI M38-A document. For amphotericin B and natamycin, the MIC was defined as the lowest drug concentration that prevented any discernible growth. For butenafine and fluconazole, the MIC was defined as the lowest concentration in which an approximately 75% reduction compared to the growth of the control was observed. Candida parapsilosis ATCC22019 was used as quality control strains to validated the results. Mean MIC and MIC range, the MIC at which 50% of the isolates tested were inhibited (MIC(50)) and the MIC at which 90% of the isolates tested were inhibited (MIC(90)), were provided for all the isolates tested by using descriptive statistical analysis with the statistical SPSS package (version 13.0). RESULTS: MIC(90) of butenafine, natamycin, amphotericin B and fluconazole were 4, 8, 2 and 512 mg/L for Fusarium spp., respectively; 0.063, 32.000, 2.000 and 256.000 mg/L for Aspergillus spp., respectively; 0.5, 8.0, 2.0 and 128.0 mg/L for Alternaria alternate, respectively; 0.125, 2.000, 0.500 and 4.000 mg/L for Curvularia lunata, respectively; and 1, 4, 1 and 256 mg/L for unusual ocular pathogens, respectively. CONCLUSIONS: Butenafine exhibits potent antifungal activity against a wide variety of ocular pathogenic fungi, especially for Aspergillus spp., Alternaria alternata, Curvularia lunata, and some unusual ocular pathogens and may have a role in future studies of antifungal eye drops and treating fungal keratitis.

In vitro comparison of the efficacies of natamycin and silver nitrate against ocular fungi.

The in vitro activity of the silver nitrate was assessed in comparison with that of natamycin against 128 corneal Fusarium isolates and 90 corneal Aspergillus isolates. MIC(90)s of silver nitrate were 2 microg/ml for Fusarium spp. and 1 microg/ml for Aspergillus spp. MIC(90)s of natamycin were 8 microg/ml for Fusarium spp. and 32 microg/ml for Aspergillus spp. Silver nitrate exhibited potent antifungal activity against ocular fungi in vitro.

Spectrum of fungal keratitis in central China.

PURPOSE: To investigate the epidemiology, risk factors, fungal spectrum and to test antifungal drug susceptibility of these isolates at a tertiary eye care referral centre in central China. METHODS: The medical and microbiology records of 2064 culture-proven cases (2064 eyes) of fungal keratitis diagnosed at Henan Eye Institute between January 2000 to March 2009 was retrospectively reviewed. The fungal isolates were identified and a subgroup of 103 isolates were subjected to drug susceptibility tests for amphotericin B, fluconazole and ketoconazole by broth microdilution method. RESULTS: A total of 2064 cases of confirmed fungal keratitis were identified. The predominant fungal species isolated was Fusarium spp. followed by Aspergillus spp. Alternaria spp. were another most common fungi in central China. Fungal keratitis was more common among men. A large proportion of the patients were middle-aged adults, and most were farmers. Ocular trauma was a highly significant risk factor and vegetative injuries were identified as a significant cause for fungal keratitis. Greatest number of cases of fungal keratitis was higher between September and December. Fusarium was mostly sensitive to amphotericin B, next to ketoconazole. Aspergillus was sensitive to amphotericin B and ketoconazole. Relatively, both Fusarium and Aspergillus were insensitive to fluconazole. CONCLUSION: Fusarium and Aspergillus are always the most isolated pathogens of fungal keratitis in central China, followed by Alternaria. Document available on the epidemiological features of a large series would greatly help ophthalmologists at primary and second health care centres in the management of this disease.

[Comparison of the activities of silver nitrate with those of three antifungal agents against ocular pathogenic fungi in vitro].

OBJECTIVE: To investigate antifungal activity of silver nitrate compared with fluconazole, ketoconazole and amphotericin B against ocular pathogenic fungi in vitro. METHODS: It was an experimental study. Susceptibility tests were performed against 260 isolates (15 genera and 29 species) of ocular pathogenic fungi by broth dilution antifungal susceptibility testing of filamentous fungi (M38-A) approved by National Committee for Clinical Laboratory Standards (NCCLS). Final concentrations ranged from 0.031 to 16.000 mg/L for silver nitrate, ketoconazole and amphotericin B, from 0.5 - 256.0 mg/L for fluconazole. Minimum inhibitory concentration (MIC) was defined as the lowest drug concentration that showed absence of growth or complete growth inhibition (100%). The end points were determined as 100% growth inhibition for silver nitrate and amphotericin B, and > or = 75% growth inhibition for ketoconazole and fluconazole. RESULTS: The MICs at which 90% of isolates were inhibited (MIC(90)) of silver nitrate, ketoconazole, amphotericin B and fluconazole were 2.000, 512.000, 32.000 and 2.000 mg/L for Fusarium species, respectively; 1.000, 256.000, 2.000 and 2.000 mg/L for Aspergillus species, respectively; 2.000, 128.000, 4.000 and 2.000 mg/L for Alternaria alternate, respectively; 2.000, 4.000, 0.125 and 0.500 mg/L for Curvularia lunata, respectively; and 1.000, 256.000, 1.000 and 1.000 mg/L for unusual ocular pathogens, respectively. Silver nitrate was highly active against Aspergillus species (92.9% susceptible at a MIC of < or = 1.0 mg/L) and Fusarium species (96.3% susceptible at a MIC of < or = 2.0 mg/L). 95.6% of Fusarium species and 90.8% of Aspergillus species exhibited resistance to fluconazole, 44.1% of Fusarium species and 42.9% of Aspergillus species exhibited resistance to amphotericin B, 66.2% of Fusarium species exhibited resistance to ketoconazole. The activity of silver nitrate against the fluconazole-resistant, ketoconazole-resistant and amphotericin B-resistant strains was high. CONCLUSION: Silver nitrate has promising activity against a wide variety of ocular pathogenic fungi in vitro, and may have a role in future studies of antifungal eye drops and treating fungal keratitis.

Histone deacetylase inhibitor attenuates experimental fungal keratitis in mice.

Fungal keratitis is one of the leading causes of blindness of infected corneal diseases, but the pathogenesis of fungal keratitis is not fully understood and therefore the treatment of the disease by medication is still under investigation. In the current study, we sought to study the effect of HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) on experimental fungal keratitis in mice. SAHA (25 mg/kg) (n = 30) or vehicle (DMSO) (n = 30) was delivered through intraperitoneal injection (IP) 24 hours after the fungal inoculation, and the same amount of SAHA injection or DMSO was followed at day 2. The expression of histone H3 (H3), acetylated histone H3 (AC-H3), histone deacetylase 1 (HDAC)1, tumor necrosis factor-α (TNFα), and Toll-like receptor 4 (TLR4) in surgically excised specimens from the patients and mice with fungal keratitis were detected by immunohistochemistry. The expression of mRNAs for Interleukin-1ß (IL-1ß), TNFα, and TLR4 were evaluated in the corneas of the mice with fungal infection and the control corneas by real-time PCR. The quantification of IL-1ß and TNFα in the corneas of the mice with fungal infection was determined by ELISA. The inhibitory effect of SAHA on mice fungal keratitis was revealed by GMS and H&E staining. We found that the downregulation of histone acetylation and upregulation of HDAC1 expression were associated with the increased inflammation response in fungal keratitis not only in humans but also in experimental animals. SAHA was able to inhibit experimental fungal keratitis in mouse by suppressing TLR4 and inflammatory cytokines such as TNFα and IL-1ß; the inhibition of HDAC may be a potential therapeutic approach for the treatment of fungal keratitis.

Identification and Characterization of Fusarium proliferatum, a New Species of Fungi that Cause Fungal Keratitis.

Fusarium proliferatum (F. proliferatum) is known as a pathogen of corn and other crops, but its role in fungal keratitis has not been well investigated. Among 877 Fusarium isolates, we identified 155 (17.7%) stains as F. proliferatum according to their morphological features and partial DNA sequencing of translation elongation factor-[Formula: see text] (EF-[Formula: see text]) in this study. In vitro antifungal susceptibility tests showed that the F. proliferatum strains were sensitive to natamycin and vorionazole but resistant to amphotericin B, fluconazol, ketoconazole and itaconazole. Most of the F. proliferatum-positive keratitis patients (44/155,28.4%) were aged 51-60 years old. The main cause of infection was injury by a plant (51/155, 32.9%). A combination of 1% amphotericin B and 3% ketoconazole cured 45.2% (14/31) and a combination of 0.5% natamycin and 0.5% voriconazole cured 59.1% (13/22) of F. proliferatum-positive patients. The date suggests that F. proliferatum identified through EF-1ɑ DNA sequencing is an important new species that causes fungal keratitis. Based on antifungal susceptibility, treatment with a combination of 0.5% natamycin and 0.5% voriconazole improves the therapeutic efficacy in F. prolifertum-positive patients.