發(fā)布時(shí)間：2018-05-19 01:40

  本文選題：外陰陰道念珠菌病 + 白念珠菌�。� 參考：《山西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:1.了解本地VVC患者念珠菌菌種分布情況及對(duì)唑類藥物的敏感性。2.探討VVC臨床分離白念珠菌ERG3、Efg1突變及表達(dá)水平與唑類藥物耐藥的關(guān)系。3.探討VVC中白念珠菌Efg1與ERG3在唑類藥物耐藥中的相互關(guān)系。方法:收集2015年11月至2016年5月就診于山西省婦幼保健院婦科門(mén)診的VVC患者陰道分泌物標(biāo)本184例,通過(guò)培養(yǎng)傳代、CHRO Magar念珠菌顯色培養(yǎng)、API 20C AUX鑒定系統(tǒng)進(jìn)行分離鑒定,共獲得146株白念珠菌。采用M27-A3微量肉湯稀釋法對(duì)其中50株白念珠菌行藥敏試驗(yàn),將其分為敏感組和耐藥組。分別提取2組菌株的基因組DNA,進(jìn)行ERG3、Efg1特異性引物擴(kuò)增后測(cè)序,通過(guò)與genbank中的標(biāo)準(zhǔn)序列進(jìn)行比對(duì),尋找其突變位點(diǎn),探討基因突變與耐藥的關(guān)系;分別提取2組菌株的總mRNA,逆轉(zhuǎn)錄為cDNA,通過(guò)熒光定量PCR(RT-PCR)測(cè)其相對(duì)表達(dá)量,分析其表達(dá)水平與耐藥的關(guān)系,并探討Efg1與ERG3在唑類藥物耐藥中的相互關(guān)系。結(jié)果:1.本試驗(yàn)共收集VVC患者陰道分泌物標(biāo)本184例,菌種分布顯示:白念珠菌146株(79.35%),光滑念珠菌22株(12.0%),熱帶念珠菌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.體外藥敏試驗(yàn)顯示:50株白念珠菌對(duì)氟康唑(fca)、伊曲康唑(itr)、伏立康唑(vrc)的耐藥率分別為44%、56%和50%,且存在交叉耐藥現(xiàn)象。3.基因測(cè)序顯示:(1)erg3測(cè)序結(jié)果:1株出現(xiàn)r352h、2株出現(xiàn)w219c,此3株菌均對(duì)fca、itr、vrc交叉耐藥。(2)efg1測(cè)序結(jié)果:6株出現(xiàn)了v86i,其中1株對(duì)itr單獨(dú)耐藥,余5株均為敏感菌株。4.耐藥組(分別耐fca、itr、vrc及交叉耐藥)和相應(yīng)敏感組的erg3、efg1mrna相對(duì)表達(dá)量統(tǒng)計(jì)學(xué)分析顯示:(1)erg3mrna表達(dá)量分析:8組數(shù)據(jù)均符合正態(tài)分布,采用t檢驗(yàn),結(jié)果用均數(shù)±標(biāo)準(zhǔn)差(?x±s)表示。(1)fca敏感組(2.01±0.60)高于耐藥組(0.59±0.36),t=10.29,p0.001,差異具有統(tǒng)計(jì)學(xué)意義;(2)itr敏感組(2.21±0.52)高于耐藥組(0.73±0.43),t=10.97,p0.001,差異具有統(tǒng)計(jì)學(xué)意義;(3)vrc敏感組(2.10±0.57)高于耐藥組(0.66±0.40),t=10.42,p0.001,差異具有統(tǒng)計(jì)學(xué)意義;(4)全敏感組(2.54±0.39)高于交叉耐藥組(0.31±0.22),t=17.99,p0.001,差異具有統(tǒng)計(jì)學(xué)意義。(2)efg1mrna表達(dá)量分析:4組耐藥組均不服從正態(tài)分布,采用秩和檢驗(yàn),結(jié)果用中位數(shù)±四分位數(shù)間距(m±qr)表示。(1)fca耐藥組(2.02±2.78)高于敏感組(0.55±0.65),平均秩為39.5和14.5,p0.001,差異具有統(tǒng)計(jì)學(xué)意義;(2)itr耐藥組(1.74±1.75)高于敏感組(0.45±0.34),平均秩為36.48和11.52,p0.001,差異具有統(tǒng)計(jì)學(xué)意義;(3)vrc耐藥組(1.88±2.22)高于敏感組(0.48±0.50),平均秩為38和13,p0.001,差異具有統(tǒng)計(jì)學(xué)意義;(4)交叉耐藥組(3.68±5.88)高于敏感組(0.34±0.11),平均秩為19.5和7.0,p0.001,差異具有統(tǒng)計(jì)學(xué)意義。(3)erg3與efg1相關(guān)性分析,采用spearman相關(guān)分析,結(jié)果顯示相關(guān)系數(shù)r=-0.614,p0.001,二者呈負(fù)相關(guān)。結(jié)論:1.本地區(qū)白念珠菌感染仍在vvc中占主導(dǎo)地位,且對(duì)唑類藥物存在不同程度耐藥,故治療vvc時(shí)應(yīng)該以菌種鑒定和藥敏分析為指導(dǎo)。2.erg3高表達(dá)可能增加白念珠菌對(duì)唑類藥物的敏感性;erg3突變可使白念珠菌對(duì)唑類藥物耐藥,w219c、r352h是否參與唑類藥物耐藥仍需進(jìn)一步的去證實(shí)。3.Efg1高表達(dá)可能增加唑類藥物的耐藥性,但Efg1突變與耐藥的關(guān)系仍需進(jìn)一步的研究。4.白念珠菌Efg1和ERG3在唑類藥物耐藥中呈負(fù)相關(guān),Efg1負(fù)性調(diào)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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.5;R711.3

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