VVC患者临床分离白念珠菌ERG3、Efg1与唑类药物耐药关系的研究

发布时间：2018-05-19 01:40

本文选题：外阴阴道念珠菌病 + 白念珠菌　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:1.了解本地VVC患者念珠菌菌种分布情况及对唑类药物的敏感性。2.探讨VVC临床分离白念珠菌ERG3、Efg1突变及表达水平与唑类药物耐药的关系。3.探讨VVC中白念珠菌Efg1与ERG3在唑类药物耐药中的相互关系。方法:收集2015年11月至2016年5月就诊于山西省妇幼保健院妇科门诊的VVC患者阴道分泌物标本184例,通过培养传代、CHRO Magar念珠菌显色培养、API 20C AUX鉴定系统进行分离鉴定,共获得146株白念珠菌。采用M27-A3微量肉汤稀释法对其中50株白念珠菌行药敏试验,将其分为敏感组和耐药组。分别提取2组菌株的基因组DNA,进行ERG3、Efg1特异性引物扩增后测序,通过与genbank中的标准序列进行比对,寻找其突变位点,探讨基因突变与耐药的关系;分别提取2组菌株的总mRNA,逆转录为cDNA,通过荧光定量PCR(RT-PCR)测其相对表达量,分析其表达水平与耐药的关系,并探讨Efg1与ERG3在唑类药物耐药中的相互关系。结果:1.本试验共收集VVC患者阴道分泌物标本184例,菌种分布显示:白念珠菌146株(79.35%),光滑念珠菌22株(12.0%),热带念珠菌6株(3.62%),近平滑念珠菌5株(2.72%),克柔念珠菌2株(1.01%),其它3株(1.63%)。2.体外药敏试验显示:50株白念珠菌对氟康唑(fca)、伊曲康唑(itr)、伏立康唑(vrc)的耐药率分别为44%、56%和50%,且存在交叉耐药现象。3.基因测序显示:(1)erg3测序结果:1株出现r352h、2株出现w219c,此3株菌均对fca、itr、vrc交叉耐药。(2)efg1测序结果:6株出现了v86i,其中1株对itr单独耐药,余5株均为敏感菌株。4.耐药组(分别耐fca、itr、vrc及交叉耐药)和相应敏感组的erg3、efg1mrna相对表达量统计学分析显示:(1)erg3mrna表达量分析:8组数据均符合正态分布,采用t检验,结果用均数±标准差(?x±s)表示。(1)fca敏感组(2.01±0.60)高于耐药组(0.59±0.36),t=10.29,p0.001,差异具有统计学意义;(2)itr敏感组(2.21±0.52)高于耐药组(0.73±0.43),t=10.97,p0.001,差异具有统计学意义;(3)vrc敏感组(2.10±0.57)高于耐药组(0.66±0.40),t=10.42,p0.001,差异具有统计学意义;(4)全敏感组(2.54±0.39)高于交叉耐药组(0.31±0.22),t=17.99,p0.001,差异具有统计学意义。(2)efg1mrna表达量分析:4组耐药组均不服从正态分布,采用秩和检验,结果用中位数±四分位数间距(m±qr)表示。(1)fca耐药组(2.02±2.78)高于敏感组(0.55±0.65),平均秩为39.5和14.5,p0.001,差异具有统计学意义;(2)itr耐药组(1.74±1.75)高于敏感组(0.45±0.34),平均秩为36.48和11.52,p0.001,差异具有统计学意义;(3)vrc耐药组(1.88±2.22)高于敏感组(0.48±0.50),平均秩为38和13,p0.001,差异具有统计学意义;(4)交叉耐药组(3.68±5.88)高于敏感组(0.34±0.11),平均秩为19.5和7.0,p0.001,差异具有统计学意义。(3)erg3与efg1相关性分析,采用spearman相关分析,结果显示相关系数r=-0.614,p0.001,二者呈负相关。结论:1.本地区白念珠菌感染仍在vvc中占主导地位,且对唑类药物存在不同程度耐药,故治疗vvc时应该以菌种鉴定和药敏分析为指导。2.erg3高表达可能增加白念珠菌对唑类药物的敏感性;erg3突变可使白念珠菌对唑类药物耐药,w219c、r352h是否参与唑类药物耐药仍需进一步的去证实。3.Efg1高表达可能增加唑类药物的耐药性,但Efg1突变与耐药的关系仍需进一步的研究。4.白念珠菌Efg1和ERG3在唑类药物耐药中呈负相关,Efg1负性调控ERG3。
[Abstract]:Objective: 1. to understand the distribution of Candida albicans in local VVC patients and sensitivity to azolic drugs.2. to explore the relationship between VVC clinical isolation of Candida albicans ERG3, Efg1 mutation and expression level and drug resistance of azolic drugs.3. to explore the relationship between Efg1 and ERG3 in the drug resistance of VVC in VVC. Methods: to collect 5 from November 2015 to 2016. 184 cases of vaginal secretions of VVC patients in gynecologic outpatient of Shanxi maternal and child health care hospital were treated in this month. 146 Candida albicans were isolated and identified by culture, CHRO Magar Candida color culture and API 20C AUX identification system. 50 strains of Candida albicans were tested by M27-A3 micro broth dilution method, and they were classified as sensitive. The genomic DNA of 2 groups of strains were extracted, respectively, and ERG3, Efg1 specific primers were amplified and sequenced. By comparing with the standard sequence in GenBank, the mutation site was searched and the relationship between the mutation and drug resistance was explored. The total mRNA of the 2 groups was extracted, and the reverse transcriptase was cDNA, and the fluorescence quantitative PCR (RT-PCR) was used to measure its phase. The relationship between the expression level and drug resistance was analyzed, and the relationship between Efg1 and ERG3 in the drug resistance of the azolics was discussed. Results: 1. a total of 184 specimens of vaginal secretions from VVC patients were collected. The distribution of bacterial species showed that Candida albicans 146 (79.35%), 22 Candida smooth Candida (12%), 6 Candida tropicalis (3.62%), and nearly 5 Candida albicans. Strains (2.72%), 2 strains of Candida korulla (1.01%) and other 3 (1.63%).2. in vitro drug sensitivity test showed that 50 strains of Candida albicans were resistant to fluconazole (FCA), itraconazole (ITR) and voriconazole (VRC) in 44%, 56% and 50%, respectively, and.3. gene sequencing showed: (1) erg3 sequencing results: 1 strains appeared r352h, 2 strain appeared w219c, this 3 strain bacteria Cross resistance to FCA, ITR, VRC. (2) efg1 sequencing results: 6 strains appeared v86i, 1 of which were resistant to ITR alone, and the remaining 5 were.4. resistant groups of sensitive strains (FCA, ITR, VRC and cross resistance) and erg3 in the corresponding sensitive group, and the relative expression of efg1mrna was analyzed: (1) the 8 groups of data were consistent with normal points. Cloth, using t test, the results were expressed with mean mean standard deviation (? X + s). (1) FCA sensitive group (2.01 + 0.60) was higher than that of drug resistant group (0.59 + 0.36), t=10.29, p0.001, the difference was statistically significant; (2) ITR sensitive group (2.21 + 0.52) was higher than the drug resistant group (0.73 + 0.43), t=10.97, p0.001, and the difference was statistically significant; (3) VRC sensitive group (2.10 + 0.57) was higher than that of drug resistance group. 66 + 0.40), t=10.42, p0.001, the difference was statistically significant; (4) the full sensitive group (2.54 + 0.39) was higher than the cross resistance group (0.31 + 0.22), t=17.99, p0.001, the difference was statistically significant. (2) efg1mrna expression analysis: 4 groups of drug resistant groups did not obey the normal distribution, using the rank sum test, the result was expressed with the median of four quantile spacing (M + QR). (1) FCA The drug resistance group (2.02 + 2.78) was higher than that of the sensitive group (0.55 + 0.65), the average rank was 39.5 and 14.5, p0.001, and the difference was statistically significant. (2) the drug resistance group (1.74 + 1.75) was higher than the sensitive group (0.45 + 0.34). The average rank was 36.48 and 11.52, p0.001, and the difference was statistically significant. The average rank of VRC resistance group was higher than that of the sensitive group. P0.001, the difference was statistically significant; (4) the cross resistance group (3.68 + 5.88) was higher than the sensitive group (0.34 + 0.11), the average rank was 19.5 and 7, p0.001, the difference was statistically significant. (3) the correlation analysis between erg3 and efg1, using Spearman correlation analysis, the results showed that the relationship was r=-0.614, p0.001, and two were negative correlation. Conclusion: 1. Candida albicans Infection is still dominant in VVC, and there are different degrees of resistance to azolic drugs, so VVC should be treated with bacterial species identification and drug sensitivity analysis to guide.2.erg3 high expression may increase the sensitivity of Candida albicans to azolic drugs; erg3 mutation can make Candida albicans resistance to azolic drugs, w219c, r352h is still involved in azolic drug resistance It is necessary to further confirm that high expression of.3.Efg1 may increase the drug resistance of azolic drugs, but the relationship between Efg1 mutation and drug resistance still needs further study of.4. Candida albicans Efg1 and ERG3 in the negative correlation of azolic drug resistance, Efg1 negative regulation of ERG3.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.5;R711.3

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