泌尿生殖道沙眼衣原体体外单独和联合药物敏感性检测
发布时间：2019-01-19 10:11
【摘要】：沙眼衣原体(Chlamydia trachomatis, Ct)感染是目前国内外最常见的性病病原体,可引起尿道炎、宫颈炎、子宫内膜炎、附睾炎、前列腺炎、不孕症多种疾病,且发病率呈逐年上升趋势,已经成为一个危害极大的公共健康问题。目前,泌尿生殖道Ct感染的发病率与其流行率密切相关,因此抗菌药物治疗是该病二级预防的重要策略；同时由于Ct疫苗至今仍不能用于临床,因此抗菌药物对于治疗Ct感染就显得更加重要。然而,泌尿生殖道Ct感染患者的临床表现常无特异性或呈隐匿性,不易引起重视,致使病情迁延、复发或反复感染,导致Ct对多种抗菌药物的敏感性降低,临床治疗失败病例急剧增加。 目的：检测泌尿生殖道沙眼衣原体标准株和临床株对阿奇霉素、米诺环素、莫西沙星、多西环素和利福平五种临床常用抗菌药物体外单独和联合药物敏感性,研究其与药物临床疗效的相关性和耐药的分子生物学机制。 方法：收集2005-2009年间到天津市性传播疾病研究所门诊就诊且符合采样标准的患者的男性尿道或女性宫颈上皮细胞,并记录其临床相关资料。采用McCoy细胞培养法对临床株进行培养,所有临床株进行五代盲传培养,对阳性标本继续传代培养,直至感染率达到90%以上,共获得41株临床菌株。对标准株和41株临床株,分别采用微量稀释法和棋盘稀释法进行五种常用抗菌药的单独和联合药敏检测。核酸扩增临床株的omp1基因并用AluⅠ、MspⅠ双酶切进行基因分型。对临床治疗失败病例的临床株,PCR方法扩增检测与大环内酯类耐药相关的23S核蛋白体RNA基因,通过产物测序检测基因突变；限制性片段分析(RFLP)检测gyrA喹诺酮耐药决定区(Quinolone-Resistance Determining Region,QRDR)常见的基因点突变。统计学方法：用SPSS11.5软件进行单因素方差分析(One-Way ANOVA),在方差分析显著的情况下,满足方差齐性要求数据两两比较采用最下显著差法即LSD (least-significant different)法进行组间多重比较。以P0.05为差异有统计学意义。 结果：总共培养临床标本238例,得到41例阳性临床株,阳性率约为17.23%。对此41株标本进行单独药敏,检测最低抑菌浓度(MIC)(单位ug/m1)分别是：阿奇霉素0.063-0.5,莫西沙星0.03-0.24,米诺环素0.008-0.064,多西环素0.063-0.5,利福平0.002-0.016。联合药敏检测部分抑菌浓度(FIC)示：阿奇霉素与莫西沙星、多西环素、利福平在体外联合时,分别对51.22%、53.66%和58.54%的菌株为协同或相加作用,拮抗作用较少；ANOVA检验示,三组之间差异无统计学意义(当a=0.05时,P=0.755)。米诺环素与阿奇霉素、莫西沙星、利福平在体外联合时,分别对90.24%、85.37%、92.68%的Ct菌株为拮抗作用,无协同作用。对临床治疗失败菌株均未检测到相应的耐药基因。 结论：1、成功分离培养Ct临床株,为进一步开展该病原体的致病机理、免疫机制、体外药物敏感性检测、耐药机制和保护性疫苗等方面的研究奠定基础。2、Ct对常用抗菌药的敏感性呈不同程度的下降,MIC值均较以往文献报道增高。对Ct体外耐药性与抗菌效果之间关系的研究并不明确。3、不同抗菌药物体外联合药敏试验,在一定程度上能够弥补单独药敏实验的一些不足,具有显著的临床意义,并且将为进一步研究抗菌药物联合作用发生机制奠定实验基础。
[Abstract]:Chlamydia trachomatis (Ct) infection is the most common venereal disease at home and abroad, which can cause urethritis, cervicitis, endometritis, epididymitis, prostatitis, and infertility. has become a public health problem that has a great danger. At present, the incidence of Ct infection in the urogenital tract is closely related to the prevalence rate, so the treatment of the antibacterial drugs is an important strategy of the secondary prevention of the disease, and because the Ct vaccine can not be used for clinical use so far, the antibacterial medicine is more important for treating the Ct infection. However, the clinical manifestation of the patients with the urinary tract Ct infection is often non-specific or latent, which is not easy to cause attention, which leads to the change of the condition, the recurrence or the repeated infection, and the sensitivity of the Ct to a plurality of antibacterial drugs is reduced, and the clinical treatment failure cases are rapidly increased. Objective: To test the sensitivity of the standard strains and clinical strains of Chlamydia trachomatis in the urogenital tract to the five clinical commonly used antibacterials in the treatment of the five clinical commonly used antibacterials, such as the azoxycycline, the minocycline, the moxifloxacin, the polycidin and the rifampin. Molecular biological machine for the study of its correlation and drug-resistance with the clinical curative effect of drugs Methods: To collect the male urethra or female cervical epithelial cells of the patients who visit and meet the sampling criteria in the outpatient department of the Institute of Sexually Transmitted Diseases in Tianjin from 2005 to 2009, and to record their clinical phase The clinical strains were cultured by the McCoy cell culture method. All the clinical strains were cultured for five generations, and the positive specimens were continued to be subcultured until the infection rate was over 90%. The standard strains and 41 clinical strains were used separately and in combination with five common antimicrobial agents by using a microdilution method and a checkerboard dilution method, respectively. Detection of the omp1 gene of the clinical strain of the nucleic acid amplification and the basis of the two-enzyme digestion of Alu I and Msp I For typing, a clinical strain of a failed case of clinical treatment was used to amplify the 23S nucleoprotein RNA gene related to the drug resistance of macrolides, and the gene mutation was detected by product sequencing; restriction fragment analysis (RFLP) was used to detect the drug resistance determining region (Quinn-Resistance Detection Reg.) of the gyrA Common genes of ion (QRDR) Point mutation. Statistical method: The single-factor analysis of variance (One-Way ANOVA) was performed with the SPSS11.5 software. In the case of the variance analysis, the most significant difference method (LSD) method was used to compare the two-to-two comparisons. Re-comparison. The difference of the difference is P0. 05. Results: In total, 238 cases of clinical specimens were cultured, and 41 positive clinical strains were obtained. The positive rate was about 1. The minimum inhibitory concentration (MIC) (unit ug/ ml) of this 41 specimens were respectively: Aichromycin 0. 063-0.5, moxifloxacin 0.03-0.24, minocycline 0. 008-0. 064, polycidin 0. 063-0.5, rifampin 0. 002-0. 016. The inhibitory concentration (FIC) of the combined drug sensitivity test part showed that, in combination with Moxifloxacin, Moxifloxacin, and rifampin in vitro, the antagonistic effect was less than that of the strains of 51. 22%, 53. 66% and 58. 54%, respectively. The VA test showed no statistical significance between the three groups (P = 0 when a = 0.05). No synergistic effect. No corresponding clinical treatment failure strains were detected Conclusion: 1. The successful isolation and culture of the Ct clinical isolates will provide the basis for further research on the pathogenesis, immune mechanism, in vitro drug sensitivity detection, drug resistance mechanism and protective vaccine of the pathogen. The MIC values were lower for different levels of decline The study of the relationship between the in vitro drug resistance and the antibacterial effect of Ct is not clear. Significant clinical significance and will be the case for further study of the combination of antibacterial drugs
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R759
本文编号：2411271
[Abstract]:Chlamydia trachomatis (Ct) infection is the most common venereal disease at home and abroad, which can cause urethritis, cervicitis, endometritis, epididymitis, prostatitis, and infertility. has become a public health problem that has a great danger. At present, the incidence of Ct infection in the urogenital tract is closely related to the prevalence rate, so the treatment of the antibacterial drugs is an important strategy of the secondary prevention of the disease, and because the Ct vaccine can not be used for clinical use so far, the antibacterial medicine is more important for treating the Ct infection. However, the clinical manifestation of the patients with the urinary tract Ct infection is often non-specific or latent, which is not easy to cause attention, which leads to the change of the condition, the recurrence or the repeated infection, and the sensitivity of the Ct to a plurality of antibacterial drugs is reduced, and the clinical treatment failure cases are rapidly increased. Objective: To test the sensitivity of the standard strains and clinical strains of Chlamydia trachomatis in the urogenital tract to the five clinical commonly used antibacterials in the treatment of the five clinical commonly used antibacterials, such as the azoxycycline, the minocycline, the moxifloxacin, the polycidin and the rifampin. Molecular biological machine for the study of its correlation and drug-resistance with the clinical curative effect of drugs Methods: To collect the male urethra or female cervical epithelial cells of the patients who visit and meet the sampling criteria in the outpatient department of the Institute of Sexually Transmitted Diseases in Tianjin from 2005 to 2009, and to record their clinical phase The clinical strains were cultured by the McCoy cell culture method. All the clinical strains were cultured for five generations, and the positive specimens were continued to be subcultured until the infection rate was over 90%. The standard strains and 41 clinical strains were used separately and in combination with five common antimicrobial agents by using a microdilution method and a checkerboard dilution method, respectively. Detection of the omp1 gene of the clinical strain of the nucleic acid amplification and the basis of the two-enzyme digestion of Alu I and Msp I For typing, a clinical strain of a failed case of clinical treatment was used to amplify the 23S nucleoprotein RNA gene related to the drug resistance of macrolides, and the gene mutation was detected by product sequencing; restriction fragment analysis (RFLP) was used to detect the drug resistance determining region (Quinn-Resistance Detection Reg.) of the gyrA Common genes of ion (QRDR) Point mutation. Statistical method: The single-factor analysis of variance (One-Way ANOVA) was performed with the SPSS11.5 software. In the case of the variance analysis, the most significant difference method (LSD) method was used to compare the two-to-two comparisons. Re-comparison. The difference of the difference is P0. 05. Results: In total, 238 cases of clinical specimens were cultured, and 41 positive clinical strains were obtained. The positive rate was about 1. The minimum inhibitory concentration (MIC) (unit ug/ ml) of this 41 specimens were respectively: Aichromycin 0. 063-0.5, moxifloxacin 0.03-0.24, minocycline 0. 008-0. 064, polycidin 0. 063-0.5, rifampin 0. 002-0. 016. The inhibitory concentration (FIC) of the combined drug sensitivity test part showed that, in combination with Moxifloxacin, Moxifloxacin, and rifampin in vitro, the antagonistic effect was less than that of the strains of 51. 22%, 53. 66% and 58. 54%, respectively. The VA test showed no statistical significance between the three groups (P = 0 when a = 0.05). No synergistic effect. No corresponding clinical treatment failure strains were detected Conclusion: 1. The successful isolation and culture of the Ct clinical isolates will provide the basis for further research on the pathogenesis, immune mechanism, in vitro drug sensitivity detection, drug resistance mechanism and protective vaccine of the pathogen. The MIC values were lower for different levels of decline The study of the relationship between the in vitro drug resistance and the antibacterial effect of Ct is not clear. Significant clinical significance and will be the case for further study of the combination of antibacterial drugs
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R759
【引证文献】
相关硕士学位论文 前1条
1 尤聪;沙眼衣原体培养阳性率提高及临床株热休克蛋白60的转录随传代的变化[D];天津医科大学;2012年
，本文编号：2411271
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