[The clinical patterns and RET proto-oncogene identification of pheochromocytoma in 13 multiple endocrine neoplasia type 2A pedigrees].

OBJECTIVE: To explore the clinical patterns and clinical significance for RET screening in adrenal pheochromocytoma (PHEO) associated with multiple endocrine neoplasia type 2A (MEN2A). METHODS: The clinical data of 32 PHEO patients with MEN2A from 13 unrelated MEN2A pedigrees from August 1989 to January 2013 were analyzed. The comprehensive medical data included systemic examinations and germline RET gene screening. RESULTS: Among 68 patients belonging to 13 MEN2A families, 32 (47.1%) presented with PHEO. There were 19 males and 13 females with a mean age of (41 ± 12) years. And the mean maximum diameter of PHEO was (4.6 ± 2.2) cm. The diagnosis of PHEO was made after medullary thyroid carcinoma (n = 12, 37.5%), simultaneously (n = 12, 37.5%), initially (n = 7, 21.9%) and death during appendectomy for PHEO-induced hypertensive crisis (n = 1, 3.1%). The diagnosis of PHEO was made before (n = 22) or after (n = 10) clinical screening. The former had 12 symptomatic cases while the latter only 1 case (12/22 vs 1/10, P = 0.024).Except for 5 asymtomatic fatal cases during non-PHEO operations, bilateral PHEO was found in 17 cases including 3 unilaterally treated cases developing another PHEO in contralateral adrenal with a lag period of 5, 10 and 17 years. There were 7 symptomatic patients in bilateral cases versus 6 in unilateral cases (7/17 vs 6/10, P = 0.440). Twenty-five patients underwent PHEO surgery: laparascopic approach in 14 cases (8 with bilateral simultaneous adrenalectomy) and open approach in 11 (2 with bilateral simultaneous adrenalectomy). And 10 patients undergoing bilateral adrenal-sparing operations or adrenalectomy required hormonal replacement therapy. During a mean observation period of 72 (1-282) months, no local recurrence, distant metastasis or Addisonian crisis were noted in 25 cases (contralateral relapse in 3 cases). Among them, 2 cases developed adrenocortical insufficiency unresponsive to an adjustment of hormonal doses.RET screening showed 4 recurrent missense substitutions in 32 MEN2A-PHEO patients: p. C634Y exon 11 (n = 27, 84.4%), p. C634R exon 11 (n = 3, 9.4%), p. C634F exon 11 (n = 1, 3.1%) and p. C618R exon 10 (n = 1, 3.1%). CONCLUSIONS: The mutations of RET proto-oncognene of PHEO in MEN2A are frequently located at codon 634. A combination of pedigree examination and RET gene screening may facilitate an early diagnosis and early treatment of asymptomatic PHEO patients in MEN2A.Laparoscopic cortical-sparing adrenalectomy for preserving adrenocortical function is a preferred surgical approach.

[Morphological characteristics in corneal smear of acanthamoeba keratitis].

OBJECTIVE: To study the morphological characteristics in corneal smear of acanthamoeba keratitis. METHODS: Experimental study. From May 1991 to December 2007, in Department of Microbiology, Beijing Institute of Ophthalmology, Beijing Tongren Hospital, 159 cases with acanthamoeba keratitis were analyzed for the laboratory results and clinical data. Giemsa's stained smear and wet-mount preparation of 159 cases were observed, all the results of cytology be photographed as the records. RESULTS: Among the 159 cases with acanthamoeba keratitis, 131 patients (82.4%) were positive on smear, and 110 cases (69.2%) were positive in culture of amoeba. The detection positive rate of smear was apparently higher than culture. Typical trophozoites, pre-encystment trophozoites, mature cysts and empty cysts could be observed by smear. So it is an important method for the clinical diagnosis. The edema and dissolved corneal epithelial cells, exudate cells, arthrospores or chlamydoconidium of mycelial fungus, vacuoles, lipid droplet and medicine crystallization should be differentiated with the pathogen. The movement of trophozoites could mainly be observed through wet-mount preparation. CONCLUSION: The detection of typical features of trophozoites, pre-encystment trophozoites, mature cysts and empty cysts by corneal smear is important for the etiological diagnosis of acanthamoeba keratitis.

[Etiological analysis of ocular non-viral microbial isolates in children and their antibiotics susceptibility in vitro].

OBJECTIVE: To analyze the types, distribution characteristics and in vitro susceptibility of antibiotic agents of ocular non-viral microbial isolates in children. METHODS: Experimental research.785 specimens obtained from children below the age of 14 in Beijing Tongren Hospital during 1999 to 2004 were cultured and identified for bacterium, fungus and Acanthamoeba respectively at Beijing Institute of Ophthalmology. Microbial culture-positive rate, susceptibility patterns and the organisms distribution of positive cultures were retrospectively analyzed. The results were statistical analyzed by chi(2). RESULTS: Of the 785 specimens, 211 were culture positive. The percentage of positive culture was 26.88%. Of those positive cultures, 176 (83.41%) had pure bacterial infections, 32 (15.17%) had pure fungal infections, and 3 (1.42%) had pure cultures of Acanthamoeba. Of the 176 bacterial positive cultures, polymicrobial infection was present in 38 cases (33 bacterial, 5 fungal and bacterial). Of 214 bacterial isolates, the most common isolate was Neisseria gonorrhoeae (39/214, 18.22%), followed by Staphylococcus epidermidis (37/214, 17.29%), and Corynebacterium spp. (36/214, 16.82%). The most common fungal pathogen isolated was Fusarium spp. representing 25.00% (8/32) of all positive fungal cultures, followed by Aspergillus spp. (15.63%, 5/32). The sensitivity of tobramycin, norfloxacin, gentamicin, amikacin, ciprofloxacin, ofloxacin, rifampicin and levofloxacin of bacteria was 62.57%, 64.94%, 70.06%, 70.87%, 71.19%, 73.89%, 85.80% and 87.50%. Gram-positive isolates were susceptible to levofloxacin. Gram-negative isolates showed high susceptible to ciprofloxacin, ofloxacin and levofloxacin. The fungal isolates were most susceptible to natamycin, followed by terbinafine, but resistant to fluconazole. CONCLUSIONS: Bacteria are the most common pathogens in non-viral microbial eye infection in children. Neisseria gonorrhoeae, Staphylococcus epidermidis, and Corynebacterium spp. are the most common bacterial pathogens, which are highly susceptible to levofloxacin and rifampicin. Fusarium spp. and Aspergillus spp. are the most common fungal pathogens, which are highly susceptible to natamycin but resistant to fluconazole.

[Mutant prevention concentrations of antibacterial agents to ocular pathogenic bacteria].

OBJECTIVE: To establish a method to measure mutant prevention concentration (MPC) in vitro, and to measure MPC of antibacterial agents for ocular bacteria caused keratitis. METHODS: It was an experimental study. Forty strains of ocular bacteria were separated from cornea in Beijing Institute of Ophthalmology, which included 8 strains of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Pseudomonas aeruginosa and Klebsiella pneumoniae respectively. The minimal inhibitory concentration (MIC) of the levofloxacin (LVF), ofloxacin (OFL), ciprofloxacin (CIP), norfloxacin (NFL), tobramycin (TOB) and chloromycetin (CHL) were determined by agar dilution method from National Committee of Clinical Laboratory Standard (NCCLS). The MPC were measured by accumulate-bacterial methods with bacterial population inoculated more than 1.2 x 10(10) colony forming units per milliliter with Mueller-Hinton broth and tryptic soy agar plate. With the software of SPSS 11.0, the datum such as the range of MIC, MPC, MIC90 and MPC90 were calculated, and the selection index (MPC90/ MI90) and mutant selection window (MSW) were obtained. RESULTS: The MI90 of LVF and TOB (4 mg/L) to Staphylococcus aureus strains were the lowest. CIP showed the lowest MIC90 (0.25 mg/L) to Pseudomonas aeruginosa among six kinds of antibacterial agents. The MIC90 of LVF to Staphylococcus epidermidis (256 mg/L), Streptococcus pneumoniae (1 mg/L) and Klebsiella pneumoniae (0.25 mg/L) were lower than other antibacterial agents. The MPC90, MSW and the MPC90/MIC90 of levofloxacin showed lower values compared with other antibacterial medicines. From all the datum, the MIC90 of CHL was the highest and the activity was the weakest. Although the activity of LVF was higher to every kind of bacteria, CIP had the highest activity antibacterial to Pseudomonas aeruginosa. The capacity of CHL and TOB was weaker than Quinolones for restricting resistant mutants on ocular bacteria. LVF had the strongest capacity for restricting resistant mutants among Quinolones. CONCLUSION: LVF has better antibacterial effects and stronger capacity for restricting the selection of resistant mutants on ocular bacteria than other antibacterial agents.

[Analysis on susceptibility of ocular bacterial pathogens to four kinds of fluoroquinolone].

OBJECTIVE: To assess the in vitro susceptibilities of ocular bacterial isolates to Gatifloxacin and other fluoroquinolones which are often used in ophthalmic treatment. METHODS: Non-randomized, non-comparative, retrospective study. Ocular bacterial isolates (n = 619) from Beijing Institute of Ophthalmology were tested for their susceptibilities to gatifloxacin, ciprofloxacin, ofloxacin and levofloxacin in vitro by the Kirby-Bauer disk diffusion method. RESULTS: The total number of ocular bacterial isolates was 619 from January, 2005 to December, 2006 including gram-positive cocci 372(60.1%), gram-negative cocci 7 (1.1%), gram-positive bacilli 60 (9.7%), gram-negative bacilli 177 (28.6%), other 3 (0.5%). The percentage of susceptibility in total ocular bacterial isolates to gatifloxacin, ciprofloxacin, ofloxacin and levofloxacin was 88.4%, 72.1%, 64.8% and 54.4%. The susceptibility of Staphylococcus species to gatifloxacin (89.9%) was significantly higher than ciprofloxacin (38.8%), ofloxacin (48.2%) and levofloxacin (61.6%). Streptococcus species showed significantly higher susceptibility to gatifloxacin (93.2%), levofloxacin (89.2%) and ofloxacin (87.8%) than ciprofloxacin (62.2%). The susceptibility of gram-positive bacilli to gatifloxacin (86.7%) was significantly higher than ciprofloxacin (58.3%); All above differences are significant (P < 0.0083). Gram-negative cocci, enteric bacilli family and Pseudomonas species have the equivalent susceptibility to gatifloxacin, ciprofloxacin, ofloxacin and levofloxacin (P > 0.05). CONCLUSIONS: Most of ocular bacterial are susceptible to gatifloxacin in vitro, especially gram-positive bacteria including Staphylococcus species, Streptococcus species and gram-positive bacilli.

The antibacterial efficacy of an aceraceous plant [Shantung maple (Acer truncatum Bunge)] may be related to inhibition of bacterial beta-oxoacyl-acyl carrier protein reductase (FabG).

Polyphenols, including flavonoids, are the major components of the extracts from aceraceous plants. They possess remarkable antibacterial and antitumour activity. Our study focused on whether the inhibition of the bacterial type II fatty acid synthesis system is the mechanism for the antibacterial effect of the related plant polyphenols. Extracts obtained from the fallen leaves of the Shantung maple (Acer truncatum Bunge) using different solvents, and the related pure compound PGG (1,2,3,4,6-penta-O-galloyl-beta-D-glucose), potently inhibited the FabG (beta-oxoacyl-ACP reductase) steps in the fatty-acid-elongation cycle with the IC(50) values between 0.9 and 7.2 microg/ml. An ethyl acetate extract appeared to inhibit FabG reductase in a mixed manner with NADPH, as did PGG with NADPH, demonstrating that they interfered with the binding of the cofactor to the enzyme. Gram-positive and Gram-negative bacteria and some fungi were used to evaluate the antibacterial abilities of different extract samples. The experiments showed that a higher polyphenol content of the extracts led to a more potent inhibitory capacity against FabG, thus enhancing the antibacterial efficacy.

[Ultrastructure study of pathogen of acanthamoeba keratitis].

OBJECTIVE: To observe the ultrastructure of the strains of acanthamoeba isolated from acanthamoeba keratitis (AK), the morphologic changes of acanthamoeba after culture with 0.02% chlorhexidine, and ultrastructure characteristics of acanthamoeba in corneal tissue of AK. METHODS: It was a experimental study. The ultrastructure of acanthamoeba strains cultured with 0.02% chlorhexidine was observed with scanning electron microscope (SEM). The excited cornea tissues from AK were observed with transmission electron microscope (TEM). RESULTS: Cultured acanthamoeba trophozoites were approximately 15 - 45 microm in diameter, appeared irregularly round or oval in shape, with rough surface and intrusion of cytoplasm. The trophozoite propagated by ways of binary division. The acanthamoeba cysts were approximately 10 - 25 microm in diameter, round in shape and with a plica-like surface. The acanthamoeba could change from trophozoite to cyst. The amoeba emerging through ostioles could turn into trophozoite and left an empty cyst. After cultured with 0.02% chlorhexidine for 24 hours, the trophozoite and cyst collapsed and distorted. However, after clinical treatment with 0.02% chlorhexidine, only the cysts could be seen in corneal tissue of AK. Ecto-and endo-cystic walls were preserved, but the cytoplasma was aggregated and the sub-cytoarchitecture were degenerated or disappeared. DISCUSSION Chlorhexidine at a concentration of 0.02% kills acanthamoeba trophozoites and cysts in vitro. Chlorhexidine (0.02%) also kills trophozoites and inhibits the activity of cysts in corneal tissues. However, it should be noticed that the inactive cysts can stay in the cornea for a long time and may cause an immuno-pathologic inflammation of the cornea.

[Experimental study of non-tuberculous mycobacterial keratitis in rabbits].

OBJECTIVE: To study clinical manifestation and pathology change of different time points of rabbit keratitis caused by non-tuberculous mycobacteria (NTM). METHODS: Forty-eight eyes from 48 rabbits were randomly divided into three groups:under-flap infection group (UFI group), under-flap infection group with corticosteroid (UFIC group) and surface infection group (SI group). The cornea infiltration area of all rabbits was observed. Bacterial quantitative culture and histopathological examination were performed. RESULTS: In the earliest stage (5 days), the cornea was cloudy and edematous. In the middle stage (7 - 14 days), cornea stromal infiltration became its major clinical sign. In the last stage (21 days), corneal neovascularization and fibrosis were developed. The mean stromal infiltration area in UFIC group was greater than those of UFI group and SI group (F = 9.362, 8.341, P < 0.05). The number of NTM in UFIC group was higher than those of UFI group and SI group (F = 411.272, P < 0.05). In histopathological examination, there were infiltrates with polymorphonuclear leukocytes (PMN) in the corneal stroma in the early stage and acid-fast bacilli was found. A plenty of lymphocytes in the cornea was found in the middle stage of NTM keratitis. In the latter stage, the fibroblast cells proliferation and corneal ulcer were cured. During the course of this infection, the number of CD4+, CD8+ T lymphocyte increased significantly, and then decreased gradually following the regress of the infection. In the stage (5 - 21 days), CD4+ T lymphocyte decreased significantly in UFIC group compared with other two groups (F = 21.907, 196.521, 12.552, 11.100, P < 0.01). CONCLUSIONS: The clinical characteristic of rabbit keratitis is the multifocal dense superficial stromal infiltrates. CD4+ T lymphocyte may play an important role in NTM keratitis.

[Etiological analysis on bacterial endophthalmitis].

OBJECTIVE: To investigate the spectrum of organisms causing bacterial endophthalmitis and their sensitivities to commonly used ocular antibiotics. METHODS: A retrospective analysis on microbiological records of 728 intraocular specimens of patients with suspected endophthalmitis from January 1999 to December 2006. Antibiotic susceptibility of these isolates was determined for commonly used ocular antibiotics using the Kirby-Bauer disk-diffusion test. RESULTS: In all, 168 organisms were isolated from 728 intraocular specimens during the study interval. Overall, 96 (57.1%) of 168 isolates were Gram-positive cocci, 52 (31.0%) were Gram-negative bacilli, 18 (10.7%) were Gram-positive bacilli, and 2 (1.2%) were Gram-negative cocci. The most common organisms identified were Staphylococcus epidermidis in 21.4% (36/168), Staphylococcus aureus in 11.3% (19/168), and Pseudomonas aeruginosa in 8.9% (15/168). For Gram-positive cocci, sensitivities were the following: vancomycin 93.4% (85/91), levofloxacin 84.4% (81/96), ofloxacin 68.8% (66/96), ciprofloxacin 59.4% (57/96), gentamicin 56.3% (54/96), and tobramycin 43.8% (42/96). For Gram-negative bacilli, sensitivities were the following: levofloxacin 88.2% (45/51), ofloxacin 76.9% (40/52), ciprofloxacin 75.0% (39/52), gentamicin 49.0% (25/51), and tobramycin 48.1% (25/52). Comparing the data from 2003 to 2006 with the data from 1999 to 2002, the susceptibility of organisms to frequently employed antibiotics was decreasing. CONCLUSIONS: The spectrum of organisms causing bacterial endophthalmitis is complex, and no single antibiotic provided coverage for all of the microbes isolated from intraocular specimens with bacterial endophthalmitis. Combination therapy is recommended as the initial empiric treatment of suspected bacterial endophthalmitis.

[Molecular characteristics of Acanthamoeba strains from keratitis patients].

OBJECTIVE: To study the molecular characteristics of Acanthamoeba strains isolated from keratitis patients. METHODS: Twenty six Acanthamoeba strains were isolated from corneal ulcers between 1994 and 2002. Partial genome DNA sequences of all strains of Acanthamoeba were amplified using PCR and then the PCR products were sequenced. The partial DNA sequences were analyzed by software Clustal X and MEGA2. The results of Acanthamoeba obtained from Beijing Tongren Hospital were compared with strains from other countries and districts. RESULTS: Of 26 Acanthamoeba strains isolated from the patients, 25 (96%) were 18S rDNA (Rns) genotype T4 and one (4%) was T3. These results were similar to those obtained from other countries and districts. CONCLUSIONS: According to the Rns genotype classification criterion, most of Acanthamoeba strains isolated from keratitis patients in Beijing are genotype T4 and only few of these strains are genotype T3. The Acanthamoeba infecting the cornea might belong to the specific 18S rDNA genotype.

Etiological analysis on ocular fungal infection in the period of 1989 - 2000.

BACKGROUND: This study was to review the distribution and shifting trend of fungal of culture specimens isolated from eyes of patients at the Beijing Institute of Ophthalmology, Tongren Hospital, Beijing, China. METHODS: The fungal culture-positive rate, the distribution and change of isolates of 2609 specimens collected in a 12-year period (1989 - 2000) were retrospectively analyzed. RESULTS: In 775 positive cultures, 707 specimens (91.2%) were from the cornea, 22 (2.8%) from the conjunctiva, 15 (1.9%) from the anterior chamber, 9 (1.2%) from the vitreous body, 3 (0.4%) from the lacrimal sac, and 19 (2.5%) from other parts of the eye. The average culture-positive rate was 29.7%. The ratio of the positive cultures in the first half year (from January to June) to those in the second half (from July to December) was 1:2.1. The main genus cultured was Fusarium sp (58.7%), followed by Aspergirum sp (16.8%). The percentage of Fusarium sp was increased from 53.6% (1989 - 1994) to 60.2% (1995 - 2000), whereas the percentage of Aspergirum sp was decreased from 22.3% (1989 - 1994) to 15.1% (1995 - 2000). CONCLUSIONS: Fusaruim sp is one of the most predominant pathogens of ocular fungal infection in northern China and its incidence tends to increase, but that of Aspergirum sp to decrease. It is very important to recognize the distribution and shifting trend of pathogenic fungi in the diagnosis, prevention and treatment of fungal keratitis.

[In vitro fluoroquinolone resistance in ocular bacterial isolates].

OBJECTIVE: To assess the in vitro resistance of ocular bacterial isolates to fluoroquinolone that are commonly used in the treatment of ocular infection. METHODS: Ocular bacterial isolates (n = 347) from Beijing Institute of Ophthalmology were tested for their resistance to ofloxacin, ciprofloxacin and norfloxacin. In vitro susceptibility testing was determined by the Kirby-Bauer disk diffusion method. RESULTS: The total number of ocular bacterial isolates was 347 from January, 1999 to December, 2000 including gram-positive cocci 192 (55.3%), gram-negative cocci 16 (4.6%), gram-positive bacilli 44 (12.7%), gram-negative bacilli 89 (25.7%), Nocardia 6 (1.7%). The percentage of resistance in total ocular bacterial isolates to ofloxacin, ciprofloxacin and norfloxacin was 28.5%, 25.9% and 34.9% respectively. The resistance of streptococcus species to ofloxacin (5.9%) and ciproloxacin (11.8%) was significantly lower than gentamycin (52.9%) and tobramycin (70.6%) (P < 0.01), and to norfloxacin (23.5%) was significantly lower than tobramycin (P < 0.01). Pseudomonas showed significantly lower resistance to ciprofloxacin (6%) ofloxacin (14.0%) and norfloxacin (14.0%) than gentamycin (42.0%) (P < 0.01 and P < 0.05). The percentage of resistance in staphylococcus species to norfloxacin (46.8%) was higher than gentamycin (30.8%) and tobramycin (27.6%) (P < 0.05 and P < 0.01 separately). The resistance of gram-positive bacilli to ofloxacin, ciproloxacin and norfloxacin. was equivalent (P > 0.05). CONCLUSIONS: The fluoroquinolones, ofloxacin and ciprofloxacin have higher overall in vitro efficacy on most of the ocular bacteria than gentamycin and tobramycin, but ofloxacin and ciprofloxacin are equivalent to gentamycin and tobramycin in efficacy against staphylococcus species.