复发性外阴阴道白假丝酵母菌病的耐药机制研究
发布时间：2018-11-06 07:32
【摘要】：目的：①对复发性外阴阴道假丝酵母菌病(Recurrent Vulvovaginal candidiasis,RWC)患者的致病白假丝酵母菌进行7种抗真菌药物的体外药物敏感实验,明确RWC致病白假丝酵母菌药物敏感性的差异；②对RVVC致病白假丝酵母菌进行药物外排泵相关蛋白-多药耐药基因MDR1、三磷酸腺苷结合转运蛋白家族CDR1、CDR2和PDR1基因表达产物的研究,明确上述耐药基因在RVVC耐药机制中的作用。 方法：①所有243株致病菌株均取自我院外阴阴道假丝酵母菌病患者,其中RVVC (实验组)致病菌株84株,VVC(对照组)致病菌株159株,均以科玛嘉显色培养基、安图显色培养基及生物梅里埃VITEK2系统鉴定明确菌种；②以FUNGUS-7真菌药敏试剂盒对已确定的213株白假丝酵母菌致病菌株进行2种多烯类药物、4种唑类药物和5-氟胞嘧啶共七种抗真菌药的体外药物敏感试验；③以荧光定量PCR (realtime fluores-cence quantitative PCR, RTFQ PCR)对已确定的敏感、中敏和耐药白假丝酵母菌菌株进行MDR1、CDR1、CDR2和PDR1基因表达产物的研究。 结果：①RVVC组白假丝酵母菌占90.48%,非白假丝酵母菌占9.52%,VVC组白假丝酵母菌占88.68%,非白假丝酵母菌占11.32%,两组白假丝酵母菌构成比没有显著差异(P0.05)；②RWC组和VVC组致病白假丝酵母菌的体外药敏结果显示：RVVC组致病白假丝酵母菌对7种抗真菌药物的敏感性依次为：制霉菌素两性霉素B(5-氟胞嘧啶益康唑咪康唑氟康唑伊曲康唑；VVC组致病白假丝酵母菌对7种抗真菌药物的敏感性依次为：两性霉素B制霉菌素5-氟胞嘧啶咪康唑益康唑氟康唑伊曲康唑；IRVVC组对制霉菌素和咪康唑的敏感性显著高于VVC组(P0.05)；③分别比较RVVC组和VVC组敏感、中敏、耐药菌株中耐药基因CDR1、CDR2、MDR1、PDR1的表达差异,结果显示：PDR1基因不是在所有菌株均有表达,RWC组PDR1基因表达率明显低于VVC组(P0.05)；PDR1基因的表达量比较显示：5-氟胞嘧啶的WC-R组的表达量高于VVC-S组和VVC-I组(P0.05),两性霉素B的VVC-R组高于VVC-I组(P0.05),伊曲康唑的RWC-R组高于WC-S组、WC-R组和RVVC-S组(P0.05);CDR1、CDR2、MDR1在所有白假丝酵母菌中均有表达,仅有CDR1基因表达量存在差异：5-氟胞嘧啶RVVC-R组明显高于WC-R组(P0.05),咪康唑中敏、耐药合并组VVC-I+R组高于VVC-S组(P0.05),氟康唑VVC-I组表达高于VVC-S组和VVC-R组(P0.05),但VVC-S组和WC-R组未检出差异(P0.05)。 结论：①复发性外阴阴道假丝酵母菌病的主要致病菌仍为白假丝酵母菌；②显色培养基适于临床上白假丝酵母菌的快速鉴定,但对非白假丝酵母鉴定准确度有限;③RVVC和VVC致病白假丝酵母菌对抗真菌药的敏感性大体一致,RVVC治疗首选制霉菌素；④PDR1、CDR1基因可能为RWC和VVC患者致病白假丝酵母菌的耐药相关基因；⑤除了耐药基因表达增加以外,RWC耐药机制的发生可能存在其它更重要相关因素。
[Abstract]:Objective: 1 to study the drug sensitivity of 7 antifungal drugs in patients with recurrent vulvovaginal Candida albicans (Recurrent Vulvovaginal candidiasis,RWC), and to determine the difference of drug sensitivity of RWC pathogenic Candida albicans. 2 the expression products of adenosine triphosphate transporter family (CDR1,CDR2) and PDR1 gene of drug efflux pump associated protein-multidrug resistance gene (MDR1,) were studied on Candida albicans caused by RVVC. To clarify the role of the above-mentioned drug resistance genes in the mechanism of drug resistance in RVVC. Methods: 1 all 243 strains of pathogenic bacteria were collected from patients with vulvovaginal candidiasis, including 159 strains of RVVC (experimental group, 84 strains of, VVC (control group). Antu chromogenic medium and biological Meridier VITEK2 system were used to identify the identified strains. (2) two polyenes, four azolides and seven 5-fluorocytosine antifungal agents were tested with FUNGUS-7 fungal susceptibility kit to 213 strains of Candida albicans. (3) the expression products of MDR1,CDR1,CDR2 and PDR1 genes were studied by fluorescence quantitative PCR (realtime fluores-cence quantitative PCR, RTFQ PCR) on the sensitive, moderately sensitive and drug-resistant Candida albicans strains. Results: in 1RVVC group, 90.48% of Candida albicans, 9.52% of non-Candida cerevisiae, 88.68% of Candida albicans, 11.32% of non-white Candida cerevisiae. There was no significant difference in the composition ratio of Candida albicans between the two groups (P0.05). In vitro antimicrobial susceptibility of pathogenic Candida albicans in 2RWC group and VVC group showed that the susceptibility of RVVC group to seven antifungal agents was nystatin amphotericin B (5-fluorocytosine econzomidazole). Conazole fluconazole itraconazole; The sensitivities of pathogenic Candida albicans to 7 antifungal agents in VVC group were amphotericin B nystatin 5 fluconazole miconazole econazole and itraconazole. The sensitivity of IRVVC group to nystatin and miconazole was significantly higher than that of VVC group (P0.05). (3) the difference of CDR1,CDR2,MDR1,PDR1 expression between RVVC group and VVC group was compared. The results showed that PDR1 gene was not expressed in all strains. The expression rate of PDR1 gene in RWC group was significantly lower than that in VVC group (P0.05). The expression of PDR1 gene in 5-fluorocytosine WC-R group was higher than that in VVC-S and VVC-I group (P0.05), and that in amphotericin B VVC-R group was higher than that in VVC-I group (P0.05). Itraconazole in RWC-R group was higher than that in WC-S group, WC-R group and RVVC-S group (P0.05). CDR1,CDR2,MDR1 was expressed in all Candida albicans, only CDR1 gene expression was different: 5-fluorocytosine RVVC-R group was significantly higher than WC-R group (P0.05), miconazole was sensitive to miconazole. VVC-I R group was higher than VVC-S group (P0.05), fluconazole VVC-I group was higher than VVC-S group and VVC-R group (P0.05), but there was no difference between VVC-S group and WC-R group (P0.05). Conclusion: 1 the main pathogenic bacteria of recurrent vulvovaginal candidiasis are still Candida albicans; (2) the chromogenic medium was suitable for rapid identification of Candida albicans in clinic, but the accuracy of identification for non-Candida albicans was limited, the sensitivity of antifungal agents against Candida albicans caused by 3RVVC and VVC was generally the same, and nystatin was the first choice in the treatment of RVVC. 4PDR1 + CDR1 gene may be the drug-resistance related gene of Candida albicans in RWC and VVC patients, and besides the increase of drug resistance gene expression, there may be other more important related factors in the mechanism of drug resistance of RWC.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.31
[Abstract]:Objective: 1 to study the drug sensitivity of 7 antifungal drugs in patients with recurrent vulvovaginal Candida albicans (Recurrent Vulvovaginal candidiasis,RWC), and to determine the difference of drug sensitivity of RWC pathogenic Candida albicans. 2 the expression products of adenosine triphosphate transporter family (CDR1,CDR2) and PDR1 gene of drug efflux pump associated protein-multidrug resistance gene (MDR1,) were studied on Candida albicans caused by RVVC. To clarify the role of the above-mentioned drug resistance genes in the mechanism of drug resistance in RVVC. Methods: 1 all 243 strains of pathogenic bacteria were collected from patients with vulvovaginal candidiasis, including 159 strains of RVVC (experimental group, 84 strains of, VVC (control group). Antu chromogenic medium and biological Meridier VITEK2 system were used to identify the identified strains. (2) two polyenes, four azolides and seven 5-fluorocytosine antifungal agents were tested with FUNGUS-7 fungal susceptibility kit to 213 strains of Candida albicans. (3) the expression products of MDR1,CDR1,CDR2 and PDR1 genes were studied by fluorescence quantitative PCR (realtime fluores-cence quantitative PCR, RTFQ PCR) on the sensitive, moderately sensitive and drug-resistant Candida albicans strains. Results: in 1RVVC group, 90.48% of Candida albicans, 9.52% of non-Candida cerevisiae, 88.68% of Candida albicans, 11.32% of non-white Candida cerevisiae. There was no significant difference in the composition ratio of Candida albicans between the two groups (P0.05). In vitro antimicrobial susceptibility of pathogenic Candida albicans in 2RWC group and VVC group showed that the susceptibility of RVVC group to seven antifungal agents was nystatin amphotericin B (5-fluorocytosine econzomidazole). Conazole fluconazole itraconazole; The sensitivities of pathogenic Candida albicans to 7 antifungal agents in VVC group were amphotericin B nystatin 5 fluconazole miconazole econazole and itraconazole. The sensitivity of IRVVC group to nystatin and miconazole was significantly higher than that of VVC group (P0.05). (3) the difference of CDR1,CDR2,MDR1,PDR1 expression between RVVC group and VVC group was compared. The results showed that PDR1 gene was not expressed in all strains. The expression rate of PDR1 gene in RWC group was significantly lower than that in VVC group (P0.05). The expression of PDR1 gene in 5-fluorocytosine WC-R group was higher than that in VVC-S and VVC-I group (P0.05), and that in amphotericin B VVC-R group was higher than that in VVC-I group (P0.05). Itraconazole in RWC-R group was higher than that in WC-S group, WC-R group and RVVC-S group (P0.05). CDR1,CDR2,MDR1 was expressed in all Candida albicans, only CDR1 gene expression was different: 5-fluorocytosine RVVC-R group was significantly higher than WC-R group (P0.05), miconazole was sensitive to miconazole. VVC-I R group was higher than VVC-S group (P0.05), fluconazole VVC-I group was higher than VVC-S group and VVC-R group (P0.05), but there was no difference between VVC-S group and WC-R group (P0.05). Conclusion: 1 the main pathogenic bacteria of recurrent vulvovaginal candidiasis are still Candida albicans; (2) the chromogenic medium was suitable for rapid identification of Candida albicans in clinic, but the accuracy of identification for non-Candida albicans was limited, the sensitivity of antifungal agents against Candida albicans caused by 3RVVC and VVC was generally the same, and nystatin was the first choice in the treatment of RVVC. 4PDR1 + CDR1 gene may be the drug-resistance related gene of Candida albicans in RWC and VVC patients, and besides the increase of drug resistance gene expression, there may be other more important related factors in the mechanism of drug resistance of RWC.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.31
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