基于25S rDNA的假丝酵母菌基因分型及抗真菌药物敏感性研究

发布时间：2018-03-09 03:36

  本文选题：外阴阴道假丝酵母菌病　切入点：复发性外阴阴道假丝酵母菌病　出处：《昆明医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的：①探讨外阴阴道假丝酵母菌病(VVC)致病菌株对7种抗真菌药物的体外实验敏感性。②探讨VVC和复发性外阴阴道假丝酵母菌病(RVVC)致病菌株基因分型与抗真菌药物最小抑菌浓度(MIC)及药物敏感性之间的价值与关联性。方法与材料：①致病菌株采集自昆明医科大学第一附属医院妇产科门诊的VVC和RVVC患者,采用VITEK(?)2、科马嘉和安图进行致病菌株的菌种鉴定。②药物敏感性检测方法为：CLSI的M27-A3,根据MIC值判断药物敏感性.③DNA提取试剂为AxyPrep Blood Genomic DNA miniprep kit,基因分型方法基于25S rDNA。④统计方法：SPSS17.0软件,采用Kruskal-Wallis H test,Mann-Whitney U test和Fisher's Exact统计方法。P0.05为差异具有统计学意义。频数表和图用于统计数据。结果：白色假丝酵母菌334例,所占比例最高,其后依次为光滑假丝酵母菌22例,热带假丝酵母菌15例,krusei假丝酵母菌12例。在VVC和RVVC致病菌株中,白色假丝酵母菌占主导地位,229株VVC菌株中,白色假丝酵母菌196株(85.6%)；155株RVVC菌株中,白色假丝酵母菌139株(89.7%)。VVC致病菌株中,非白色假丝酵母菌中,光滑假丝酵母菌(7.4%),热带假丝酵母菌(3.9%)、克柔假丝酵母菌(3.1%).而在RVVC致病菌株中,热带假丝酵母菌(3.9%)、光滑假丝酵母菌3.2%,克柔假丝酵母菌(3.2%).白色假丝酵母菌致病菌菌株在妊娠和非妊娠患者VVC致病菌株中亦占主导地位,分别为(88.7%)和(87.0%),在非妊娠组中,非白色假丝酵母菌致病菌株光滑假丝酵母菌(6.8%),热带假丝酵母菌(4.0%)和克柔假丝酵母菌(2.2%),相反,妊娠组中非白色假丝酵母菌致病菌株主要为克柔假丝酵母菌和热带假丝酵母菌(3.2%),无光滑假丝酵母菌致病菌株。药物敏感性结果显示,85.4%(286/335)白色假丝酵母菌致病菌株对氟康唑敏感,而仅有28.6%(14/49)非白色假丝酵母菌致病菌株对氟康唑敏感,两组差异具有统计学意义(P0.001)。同样地,76.1%(255/335)白色假丝酵母菌致病菌株对克霉唑敏感,61.2%(30/49)非白色假丝酵母菌致病菌株对克霉唑敏感,两组差异具有统计学意义(P=0.011)。相反,非白色假丝酵母菌致病菌株对咪康唑更敏感(P0.001,95.9%(47/49)非白色假丝酵母菌致病菌株对咪康唑敏感,而仅72.5%(243/335)白色假丝酵母菌致病菌株对咪康唑敏感。非白色假丝酵母菌致病菌株对伊曲康唑63.3%(31/49)的敏感性也较白色假丝酵母菌致病菌35.2%(118/335)株高,非白色假丝酵母菌致病菌株对氟康唑也显示了不同的药物敏感性,93.3%(14/15)热带假丝酵母菌致病菌株较光滑假丝酵母菌致病菌株敏感,差异具有统计学意义(P0.001)。主要的三种非白色假丝酵母菌致病菌株对5-氟胞嘧啶、咪康唑、两性霉素B敏感。在WC和RVVC两组比较中,VVC组对氟康唑的敏感性高于RVVC组, (P=0.04)分别88.8%(174/335)和80.6%(112/335)。然而,VVC组对咪康唑(P=0.001)和两性霉素B(P=0.007)的敏感性低于RVVC组,5-氟胞嘧啶和克霉唑在两组中均显示了较高的敏感性,大于80%的致病菌菌株对这两种药物敏感。相反,制霉菌素和伊曲康唑在两组均敏感性下降,耐药率大于40%。在妊娠组中,VVC致病菌株对咪康唑(P0.001)和两性霉素(P=0.028)敏感较非妊娠组高,RVVC致病菌菌株对MCZ(P=0.018),ITR(P=0.013)和NYS(P=0.006)较非妊娠组敏感。335株致病菌株中,309株(92.2%)鉴定为基因型A(450 bp),19株(5.6%)鉴定为基因型B(840 bp),7株(2%)鉴定为基因型C (450 bp and 840 bp),本研究中无片段大小为1040 bp和1080 bp的与都柏林菌属相关PCR产物。基因型A在VVC和RVVC组所占比例均高,两组比较无统计学差异, (P=0.061)。196株VVC致病菌株,89.8%(176/196)为基因型A,10.2%(20/196)为基因型B和C。 139株RVVC致病菌株中,95.7%(133/139)为基因型A,4.3%(6/139)为基因型B和C。非白色假丝酵母菌致病菌菌株中,除1株光滑和2株克柔假丝酵母菌为基因型B外,余均为基因A。分析基因型与抗真菌药物敏感性相关性,基因型B和C(16／26,61.5%)对伊曲康唑的敏感性高于基因型A(103/309,33.3%),(P=0.003)。所有包含840bpPCR产物以及三种非白色酵母菌都对5-氟胞嘧啶敏感。基因型B和C的5-氟胞嘧啶和伊曲康唑的MIC值要低于基因型A,两组差异具有统计学意义,分别为(P=0.008)和(P=0.003)。基因型A的最低MIC值抗真菌药物是克霉唑和两性霉素B,(MIC90=1μg/ml),而基因型B和C的最低MIC值抗真菌药物是5-氟胞嘧啶,(MIC90=0.475μg/ml)。基因型A的最高MIC值抗真菌药物是制霉菌素、咪康唑和伊曲康唑(MIC90=16μg/ml),而基因型B和C的最高MIC值抗真菌药物是制霉菌素(MIC90=10.4μg/ml)。结论：①白色假丝酵母菌致病菌株是WC和RVVC妊娠或非妊娠患者最常见的致病菌株。②氟康唑和克霉唑对非白色假丝酵母菌致病菌菌株的作用效果欠佳,因其体外实验中显示了比白色假丝酵母菌致病菌株更高的耐药性。然而,非白色假丝酵母菌致病菌株显示了对咪康唑及伊曲康唑的药物敏感性高于白色假丝酵母菌致病菌株。③VVC和RVVC组中,非白色假丝酵母菌致病菌株未发现不同药物的敏感性差异。RVVC组白色假丝酵母菌致病菌株对氟康唑的敏感性低于VVC组,这警示我们未来对该药物的临床应用。另一方面,RVVC组白色假丝酵母菌致病菌株对咪康唑和两性霉素B的敏感性高于WC组。④非妊娠期致病菌株对制霉菌素、两性霉素B、咪康唑和伊曲康唑的药物敏感性要高于妊娠期致病菌株。除伊曲康唑外,所有的唑类药物在妊娠期致病菌株中均显示敏感。⑤基因型A在所有的致病菌株中占主导地位,其后依次为基因型B和C。基因型无VVC和RVVC无相关性,但对药物的MIC值及药物敏感性有一定影响。表达1组内含子的致病菌株可能是对5-氟胞嘧啶药物敏感性的主要因素,因所有含840 bp PCR产物的白色和非白色假丝酵母菌致病菌株对其敏感。
[Abstract]:Objective: To investigate the vulvovaginal candidiasis (VVC) in vitro sensitivity of pathogenic strains to 7 kinds of antifungal drugs. To explore VVC and recurrent vulvovaginal candidiasis (RVVC) pathogenic strains of genotype and antifungal drugs of minimal inhibitory concentration (MIC) value and the relationship between drugs and sensitivity. Materials and methods: the pathogenic strains collected from the outpatient department of gynecology and obstetrics of the First Affiliated Hospital of Kunming Medical University and VVC RVVC patients with VITEK (?) 2, Antu Jiahe Tacoma identification of pathogenic strains. The drug sensitivity detection method: CLSI M27-A3, according to the MIC value to determine drug sensitivity. The DNA extraction reagent AxyPrep Blood Genomic DNA miniprep kit 25S rDNA., the statistical method based on genotyping methods: using Kruskal-Wallis SPSS17.0 software, H test, Mann-Whitney U test and Fisher's Exact statistics .P0.05 was a statistically significant difference. The frequency table and chart for statistical data. Results: Candida albicans in 334 cases, the highest proportion, followed by Candida glabrata in 22 cases, 15 cases of Candida tropicalis, 12 cases of krusei Candida spp. in VVC and RVVC pathogenic strains of Candida. Albicans is dominant, 229 VVC strains, 196 strains of Candida albicans (85.6%); 155 RVVC strains, 139 strains of Candida albicans (89.7%).VVC in non pathogenic strains, Candida albicans, Candida glabrata (7.4%), Candida tropicalis (3.9%), Candida krusei (3.1%). While in RVVC pathogenic strains of Candida tropicalis (3.9%), 3.2% Candida glabrata, Candida krusei (3.2%). Candida albicans strains of pathogenic bacteria are dominant in pregnant and non pregnant patients with VVC isolates, respectively. For (88.7%) and (87%), in the non pregnant group, non Candida albicans strains of Candida glabrata (6.8%), Candida tropicalis (4%) and Candida krusei (2.2%), on the contrary, in the non pregnant group of Candida albicans strains for c.krusei Candida albicans and Candida tropicalis (3.2%), non Candida pathogenic strains. The drug sensitivity results showed that 85.4% (286/335) of Candida albicans strains sensitive to fluconazole, and only 28.6% (14/49) non Candida albicans strains to fluconazole sensitive, with significant difference between two groups (P0.001) similarly, 76.1% (255/335) of Candida albicans strains sensitive to clotrimazole, 61.2% (30/49) non Candida albicans strains sensitive to clotrimazole, the difference between the two groups had statistical significance (P=0.011). On the contrary, non Candida albicans induced Isolates of miconazole (P0.001,95.9% (47/49) is more sensitive to non Candida albicans strains sensitive to miconazole, while only 72.5% (243/335) of Candida albicans strains sensitive to miconazole. Non Candida albicans strains of itraconazole in 63.3% (31/49) is the sensitivity of Candida yeast 35.2% pathogenic strains (118/335), non Candida albicans strains also showed different sensitivity to fluconazole, 93.3% (14/15) of Candida tropicalis strains were Candida strains sensitive, the difference was statistically significant (P0.001). Three non pathogenic strains of Candida albicans the main 5- of flucytosine, miconazole and amphotericin B in WC and RVVC sensitive. The comparison between the two groups, VVC group of sensitivity to fluconazole was higher than that of group RVVC (P=0.04), 88.8% (174 /335) and 80.6% (112/335). However, VVC group 瀵瑰挭搴峰攽(P=0.001)鍜屼袱鎬ч湁绱燘(P=0.007)鐨勬晱鎰熸，

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