VVC患者临床分离白念珠菌white-gray-opaque三稳态转换对其毒力活性的影响

发布时间：2018-01-17 17:16

  本文关键词：VVC患者临床分离白念珠菌white-gray-opaque三稳态转换对其毒力活性的影响　出处：《山西医科大学》2016年硕士论文　论文类型：学位论文

  更多相关文章： 外阴阴道念珠菌病 白念珠菌 表型转换 Sap分泌活性

【摘要】：目的1、了解本地区VVC患者念珠菌的菌种分布情况及对唑类药物的敏感性;2、通过YPD培养基、Lee,s葡糖糖培养基、Lee,s N-乙酰葡糖胺(Glc NAC)培养基、CO2及温度来诱导white、gray、opaque三种不同表型细胞的产生。3、利用牛血清白蛋白培养基(BSA)检测white、gray、opaque三种不同表型细胞分泌型天冬氨酸蛋白酶(Sap)的活性差异。方法纳入2015年2月至2015年10月就诊于本科门诊,疑似VVC患者阴道分泌物标本252例,并通过10%KOH镜检、沙堡弱培养基培养、CHROMagar念珠菌显色培养、API20CAUX鉴定系统进行培养分离鉴定,初步获得64株白念珠菌,采用ATB Fungus3酵母菌样真菌敏感性检测系统对其进行药敏试验。提取64株白念珠菌的基因组DNA,真菌通用引物进行PCR扩增,分子生物学方法进一步检测其是否为白念珠菌。利用MTLa1、MTLɑ2特异性引物,筛选出MTL纯合型菌株。先利用YPD培养基对其进行表型初筛,再利用温度、CO2含量的不同、Lee's葡萄糖和Lee's N-乙酰葡糖胺(Glc NAC)培养基来诱导其他两种表型细胞的产生,最后利用BSA培养基检测每株菌株三种不同表型细胞Sap活性大小,从而反应白念珠菌的表型转换对其毒力活性的影响。结果(1)共收集VVC患者阴道分泌物252份,真菌培养阳性者154例,阳性率为61.1%。其中白念珠菌64株(41.6%),热带念珠菌31株(20.1%),光滑念珠菌22株(14.3%),近平滑念珠菌8株(5.2%),克柔念珠菌5株(3.2%),其它24株(15.6%)。(2)64株白念珠菌药敏试验结果:白念珠菌对5-氟胞嘧啶和两性霉素B有较高的敏感性,对氟康唑、伊曲康唑、伏立康唑的耐药率分别为39.01%、40.63%和45.31%,存在交叉耐药现象。(3)64株菌株的MTL a、ɑ基因筛查显示只有3株为纯合型菌株(F22为a/a型,F10、F48为ɑ/ɑ型),余均为a/ɑ杂合型。(4)64株菌株中只有3株纯合型菌株成功进行了表型转换,诱导出white、gray和opaque三种不同表型细胞。(5)3株菌株white、gray、opaque细胞Sap分泌活性存在差异,其活性大小依次为gray、opaque、white。结论1、本地区VVC患者所感染的致病真菌仍以白念珠菌为主,对FCA耐药率相对较低,可作为临床治疗的一线用药。2、利用不同的培养基、温度、不同CO2浓度,可以成功诱导MTL纯合型菌株进行表型转换,得到white、gray、opaque三种细胞。3、VVC患者分离出的白念珠菌中三种不同表型细胞的毒力grayopaquewhite。
[Abstract]:Objective to understand the distribution of 1 species of local VVC in Candida and sensitivity to azole drugs; 2, by YPD medium, Lee medium, s glucose, Lee, s N- acetylglucosamine (Glc NAC) medium, CO2 and temperature to induce white, gray, opaque three different the phenotype of cells produce.3 culture medium using bovine serum albumin (BSA) detection of white, gray, opaque three different types of cells secreted aspartic proteinase (Sap) activity difference. Methods included in February 2015 to October 2015 in the outpatient clinic, patients with suspected VVC vaginal secretion samples of 252 cases, and through the 10%KOH examination sand castle, weak medium, CHROMagar Candida culture, API20CAUX identification system culture isolation and identification, obtained 64 strains of Candida albicans, drug sensitivity tests were performed using ATB Fungus3 yeast like fungi susceptibility detection system from 64 strains of Candida albicans. The genomic DNA of bacteria and fungi universal primers for PCR amplification, molecular biology method to detect whether it is Candida albicans. The use of MTLa1, MTL. 2 primers were screened for MTL homozygous strains. Using YPD culture medium on the phenotypic screening, using temperature, different content of CO2, Lee's glucose Lee's and N- acetylglucosamine (Glc NAC) to induce the other two cell phenotype medium, finally using BSA medium to detect each of three strains of different size and activity of Sap cell phenotype, response phenotype of Candida albicans virulence conversion effect on its activity. Results (1) were collected in patients with vaginal VVC the secretion of 252 copies, 154 cases of fungal culture positive, the positive rate of 61.1%. among 64 strains of Candida albicans (41.6%), Candida tropicalis 31 strains (20.1%), 22 (14.3%) strains of Candida glabrata, Candida parapsilosis (5.2%), 8 strains of Candida krusei 5 strains (3.2%), the 瀹，

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