Objective:To explore the effects of laser irradiation parameters (irradiation frequency and single duration) on tear secretion, lens and retina.Methods:Thirty-six healthy guinea pigs were randomly divided into 6 groups with random number table method according to different frequency and single exposure duration of laser to the eye, namely, high frequency short time (HFST) group, high frequency long time (HFLT) group, medium frequency short time (MFST) group, medium frequency long time (MFLT) group, low frequency short time (LFST) group and low frequency long time (LFLT) group, 6 for each group.The right eyes were irradiated with 500 lx laser as experimental eyes, and the left eyes of the guinea pigs served as the control eyes.The high, medium and low irradiation frequencies were defined as 15 times, 10 times and 5 times, respectively, and the short and long period was defined as 30 seconds and 60 seconds each time, respectively.The right eyes were irradiated based on the grouping at a 10-minute interval.The tear secretion was detected by SchirmerⅠtest; lens opacity was assessed under the slit-lamp microscope; fundus photography was performed to evaluate the general morphology of retina; retinal function was evaluated by electroretinogram (ERG) record and the thickness of retinal outer nuclear layer was measured by histopathology examination.This study protocol was approved by the Medical Ethics Committee of Air Force Military Medical University (No.20181203), and the use and care of the experimental animals complied with the ARVO statement.Results:The tear secretion was 8.00(7.37, 9.00), 8.75(8.25, 9.00), 8.50(7.75, 9.50), 9.00(8.50, 9.50), 8.00(7.37, 8.75) and 8.25(7.75, 8.75) mm/5 min in the HFST group, HFLT group, MFST group, MFLT group, LFST group and LFLT group, respectively, without significant difference among the groups(χ 2=5.502, P=0.240); after laser irradiation, there were no statistically significant differences in tear secretion between the control eyes and laser-irradiated eyes in all the groups (all at P>0.05). The lenses were clear and the fundus was normal through the experimental duration in all the groups.The amplitude of ERG a-wave was significantly reduced in the HFST group in comparison with the LFST group (P<0.05), and there was no significant difference in the b-wave amplitude among the six groups (F=1.358, P=0.268). The ERG a-, b-wave amplitudes were not significantly different between the control eyes and laser-irradiated eyes in various groups (both at P>0.05). There was no significant difference in the thickness of the outer nuclear layer of retina among the HFST group, HFLT group, MFST group, MFLT group, LFST group and LFLT group (F=0.952, P=0.463). Conclusions:The 500 lx laser irradiation is safe to ocular surface and lens, but there are some injuries to retinal function, and the injury degree is related to laser irradiation frequency.

Objective To analyze the management and outcomes of patients with ST-segment elevation myocardial infarction (STEMI) in Liaoning province.Methods The data were collected from a prospective and multicenter registry study including 8 tertiary hospitals and 12 secondary hospitals in Liaoning province.Total 1429 patients with acute STEMI admitted to hospitals from June 2009 to June 2010 were included in the study.A unified follow-up questionnaire was applied on patient discharged.Results The average age of patients was (63 ± 13)years.37.4％ of patients recognized the disease as heart disease and 39.7％ were transported by emergency ambulance with a median symptom-to-door time of 150 min.52.9％ patients underwent emergency reperfusion therapy,including fibrinolytic therapy (24.4％) and primary percutaneous coronary intervention (PCI,28.1％).The in-hospital treatment included aspirin (99.6％),clopidogrel (81.9％),statins (90.1％),low molecular weight heparin (89.5％),β-blocker (66.0％),angiotensin converting enzyme inhibitor (ACEI)/ angiotensin receptor blocker (ARB)(66.6％).The in-hospital mortality was 10.7％ ; the mortality in females was higher than that in males (18.3％ vs.7.9％,P ＜ 0.01) and the mortality in older patients (≥ 65 years) was higher than that in younger patients (＜65 years)(17.0％ vs.5.2％,P ＜0.01).The follow-up treatment included:aspirin (81.1％),clopidogrel (45.0％),statins (61.0％),β-blocker (48.3％),ACEI/ARB (42.4％).The follow-up mortality was 5.0％ after hospital discharge.Conclusions Longer pre-hospital delay is commonly seen in STEMI patients.There is still certain gap of emergency reperfusion therapy and the evidence-based medication with related clinical guidelines of STEMI management in Liaoning.

Objective To analyze the composition and distribution of pathogens from 1366 superficial candidiasis cases in Shanghai.Methods Candida species identification was carried out for 1366 adults or children with superficial candidiasis by using CHROMagar Candida plates,API20C AUX system,etc.Pal's agar,Xylose assimilation and the test for growth at 45 ℃ were utilized to differentiate Candida dubliniensis.Newly identified pathogenic Candida species including Candida orthopsilosis,Candida metapsilosis,Candida fermentati,Candida nivariensis and Candida bracarensis were differentiated by molecular biological methods.Finally,the composition and distribution of pathogens in superficial candidiasis cases were statistically analyzed.Results A total of 1366 Candida strains,included 2 Candida orthopsilosis strains and 4 Candida metapsilosis strains,were isolated from these cases.Among these isolates,Candida albicans predominated(79.0％),followed by Candida parapsilosis(9.5％),Candida tropicalis(2.9％)and Candida guilliermondii(1.9％).The composition of Candida species was significantly different between child and adult patients(x2 =196.46,P ＜ 0.01),with the isolation rate of non-albicans Candida species being 14.4％ and 45.8％ respectively in child and adult patients.Conclusions Candida albicans is still the dominant pathogen of superficial candidiasis.Candida orthopsilosis and Candida metapsilosis can cause superficial candidiasis.The isolation rote of non-albicans Candida species is higher in adult patients than in child patients.

Objective To establish a multiplex PCR targeting the intergenic spacer regions (IGS) for the identification of Cryptococcus neoformans var.grubii,var.neoformans and Cryptococcus gattii.Methods Primers were designed by using the software ClustalW2 and Oligo 6 based on the sequence of IGS1 region,which shows high sequence variability in the genome of Cryptococcus neoformans and Cryptococcus gattii.,for the multiplex PCR.Then,the developed multiplex PCR was performed to identify 51 Cryptococcus neoformans strains representing genotypes VNI-VNIV and VNB as well as 41 Cryptococcus gattii strains representing genotypes VGI-VGIV.The identification results were compared with those from common PCR by using primers GPA1A,CLA4D and SODlgattii specific to Cryptococcus neoformans var.grubii,var.neoformans and Cryptococcus gattii,respectively,as well as with those from the canavanine-glycine-bromothymol blue (CGB) medium-based culture.Results The developed multiplex PCR successfully identified the 92 Cryptococcus neoformans and Cryptococcus gattii.strains,and yielded negative results from the other tested pathogenic yeasts,which revealed a high specificity of the designed primers.False positive results were observed in the identification of two Cryptococcus gattii strains with GPA1A primer-based PCR,one Cryptococcus gattii strain with CLA4D primer-based PCR,one var.grubii strain and one var.neoformans strain with CGB culture,while no false negative results were observed in the detection of these Cryptococcus strains by any of these methods.Conclusions The developed multiplex PCR in this study can rapidly and accurately identify Cryptococcus neoformans var.grubii,var.neoformans,AD hybrid,and Cryptococcus gattii,with superior performance in comparison with common PCR and CGB medium-based culture.

Objective To establish a PCR method for rapid identification and sequence typing of VG Ⅱ allele of the intergenic spacer region (IGS) of Cryptococcus gattii.Methods Since IGS1 was of high sequence variation,multiple alignments were conducted by ClustalX 2 in IGS1 of Cryptococcus gattii and Cryptococcus neoformans,and then primer sets specific to genotype VG Ⅱ was designed for PCR analysis.The specificity of the primer pair was detected by amplification of the other genotypes in Cryptococcus gattii,Cryptococcus neoformans,and other pathogenic yeasts.The amplified fragments from VG Ⅱ genotype were sequenced and subtyped.Results Using the PCR analysis developed in this study,all VG Ⅱ genotype strains tested were amplified,whereas no amplification was obtained from other genotypes or yeast species involved herein.Three polymorphic nucleotide sites at 72,79 and 104 bp in the fragment amplified could be used to distinguish sub-genotypes within VG Ⅱ genotype.Conclusions The PCR analysis developed in this study can be used for rapid identification of genotype VG Ⅱ of Cryptococcus gattii.The sequence typing based on the amplified fragment from IGS1 may be performed for screening the highly virulent sub-genotype VGⅡ a.

Objective To assess the subgenotypes of pathogenic Cryptococcus gattii isolates from China and to elucidate the epidemiological links between these domestic isolates and those from other parts of the world. Methods DNA was extracted from 9 clinical isolates of Ctyptococcus gattii from China. The partially variable regions of the three unlinked loci, namely IGS1, PLB1 and GEF1, were amplified and sequenced, and the bioinformation at these loci was obtained from GenBank for multi-locus sequences alignment and phylogenetic analysis. Results Of these 9 clinical isolates, 8 were genotype VG Ⅰ and mating type α with the same sequences at the tested regions as the reference strain WM276, which was a representative isolate of an independent subgenotype; 1 was of genotype VG Ⅱ and mating type α, which was the first report in China, with the tested sequences consistent with those of the referrence strain R272. Sequencing and phylogenetic analysis of GEF1 gene, which was located at mating type locus, successfully identified the genotypes and mating types of all the Cryptococcus gattii isolates involved here. Conclusions Multi-locus sequence analysis shows that causative Cryptococcus gattii isolates of genotype VG Ⅰ in China carry similar sequences at the tested loci in IGS1, PLB1 and GEF1 genes, to a widely distributed subgenotype in the world, and the sequences of the first VG Ⅱ genotype isolate from China resemble the less virulent subgenotype VG Ⅱ b found in Vancouver islands.

Objective To identify the AD hybrid strains and its hybrid types within Cryptococcus neoformans.Methods Difierent hybrid types of AD strains were analyzed by PCR 0f STE20 and MF genes within MAT locus and CIA4 and GPal genes out of MAT locus.The PCR-RFLP analysis of g6341 gene was also performed.Results The mating types of 18 AD strains were precisely identified by PCR of STE20 gene,whereas those of H strain were not identified.CL44 gene was better than the GPal gene in PCR identification of the AD hybrids.In the RFLP analysis of g6341 gene,AD strains were grouped into 2 distinct RFLP patterns based on the mating type on serotype A allele.The mating types of AD strains were not identified by the molecular analyses based on the CL44,GPal and g6341 genes.Conclusion It is necessary to use multi-locus analyses of genes within and out of the MAT locus in precise identification of the AD strains and their hybrid types of Cryptococcus neoformans.

Objective To determine in vitro drug susceptibility to five antifungal agents of clinical Cryptococcus neoformans strains isolated from different areas of China in recent ten years. Methods Eighty clinical isolates of Cryptococcus neoformans were isolated from Shanghai, Guangdong, Fujian, Beijing and some other areas of China from 1998 to 2007. The minimal inhibitory concentrations (MIC) of the isolates to five antifungal agents, including amphotericin B, fluconazole, flucytosine, itraconazole and voriconazole, were determined using broth microdilution procedure (document M27-A2) recommended by the Clinical and Laboratory Standards Institute (CLSI). Kruskal-Wallis rank sum test was employed for the statistical analysis. Results The MIC50 of the Cryptococcus neoforrnans isolates tested for amphotericin B, fluconazole, flucytosine, itraconazole and voriconazole were 0.5, 4, 2, 0.25 and ≤0.031 3 mg/L, respectively; and the MIC<,90> of the isolates tested for the above antifungal agents were 1, 8, 4, 0.5 and 0.062 5 mg/L, respectively. Among the tested isolates, 3 (3.8 %) were resistant to flucytosine, 4 (5.0 %) were resistant to itraconazole. All isolates were susceptible to amphotericin B and voriconazole. There was no significant difference in MIC of the strains isolated from any particular years to the five agents (χ2=0.500,2.687,2.211, 2.660,0.677,P>0.05). Conclusions The Cryptococcusneoformans isolates are highly susceptible to the five antifungal agents, while a few strains are resistant to flucytosine or itraconazole. The drug susceptibilities of the strains isolated from particular years are similar.

Objective To evaluate the efficiency of PCR-restriction fragment length polymorphism (PCR-RFLP)aiming at the structure gene g6341,versus PCR fingerprinting analysis in the genotyping of Cryptococcus neoformans MethodsEight reference strains and 68 clinical and environmental isolates of C.neoformans were genotyped by PCR-RFLP and PCR fingerprinfing.In PCR fingerprinting,the minisatel lite-specific core sequence of wild-type phage M13 was used as a single primer.The structure gene g6341 was selected for PCR-RFLP analysis by sequence alignments of multiple genes,a pair of pnmers were developed based on the conserved region of g6341 gene.PCR products were digested with the appropriate restriction endonucleases,and RFLP profiles were analyzed.Partial sequence analysis of g6341 gene was performed for different genotypes of C.Neoformans.Phylogenetic analysis was done to study the relatedness between these genotypes.Results As sequence homology analysis showed,g6341 gene was suitable for RFLP analysis.In the case of enotyping of 76 C. Neoformans strains,the results obtained from PCR-RFLP were consistent with those from PCR fingerprinting.Sequence analysis of g6341 gene revealed a homology of 84%-97%among the eight genotypes as well as a consistency of 99%-100%within a same genotype.In the phylogenetic tree,genotypes VNⅠ,VNⅡ,VNⅢand VNⅣ belonged to one cluster,and genotypes VGⅠ,VGⅡ,VGⅢ and VGⅣ to another cluster.Conclusions PCR-RFLP analysis aiming at the structure gene g6341 is a useful tool to genotype C.neoformans.Sequence analysis of g6341 gene can disclose the relatedness among different molecular types of C.neoformans.

Objective To evaluate the role of Restriction fragment length polymorphism (RFLP) analysis in detection of the fragment of GEF1α/a gene which are both located at ct and a mating type loci in identification of species, varieties, genotypes and mating types of Cryptococcus neoformans species complex(Cryptococcus neoformans and Cryptococcus gattii). Methods The GEF1α/a gene was selected from 20 genes which both located at α and a mating type loci for RFLP analysis, according to the requirements of sequence similarities and primer design in PCR-RFLP analysis. Primer pair was designed from the conserved regions of GEF1α/a genes of distinct genotypes and mating types of reference strains to amplify a fragment of GEF1α/a gene from Cryptococcus neoformans and Cryptococcns gattii strains tested. Sequence alignment,restriction maps analysis, endonucleases selection and electrophoresis stimulation were conducted by using DNAMAN and Vector NTI software. EeoT14 Ⅰ and Hap Ⅱ endonucleases were selected for RFLP analysis of the GEF1α/a fragments amplified from 125 isolates of Cryptococcns neoformans and Cryptococcus gattii. Results An approximate 1 300 bp fragment was amplified from total 82 Cryptococcus neoformans and 43 Cryptoceccus gattii isolates. However, negative PCR results were found in the reference strains of Cryptococcus laurentii, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krnsei,Candida glabrata, Trichosporon asahii, Aspergillus fumigatns and Aspergillus flavus. RFLP analysis successfully identified the species, varieties, genotypes and mating types of total 125 isolates of Cryptococcus neoformans and Cryptococcns gattii tested in this study. Condusion PCR-RFLP analysis of the GEF1α/a fragment has the potential value in identification of species, varieties, genotypes and mating types of Cryptococeus neoformans species complex simultaneously and rapidly, and may be a useful tool in molecular epidemiological analysis.