【作者】 杨丽娜；

【导师】 刘全忠； 齐蔓莉；

【作者基本信息】 天津医科大学 ， 皮肤病与性病学， 2008， 硕士

【摘要】 沙眼衣原体（Chlamydia trachomatis,Ct）是一种严格的细胞内寄生的微生物,感染人体后可以引起多种疾病。泌尿生殖道的Ct感染,除引起非淋菌性尿道炎（non-gonococcal urethritiS,NGU）外,还可引起多种并发症并给患者带来严重的心理负担。近年来,性传播疾病的发病率在全世界范围内迅猛增加,其中Ct所致的NGU已经成为最常见的性传播疾病之一。据世界卫生组织估计,每年有9300万新发病例,给社会造成严重的经济负担。泌尿生殖道Ct感染的发病率在我国有逐年增加的趋势,已成为危害公共健康的一大问题。治疗Ct感染的主要药物是抗菌药,以往认为抗菌药有超过90%的治愈率,但目前临床治疗Ct感染失败的报道愈来愈多。治疗失败不仅与病人年龄、治疗期间性交活动导致再感染有关,而且也和患者依从性、药物生物利用度、Ct载量等因素有关。而更为重要的是临床株中耐药菌株常导致治疗失败,甚至形成持续感染状态,造成感染长期不愈。随着抗菌药的不断更替和合理使用的加强,Ct对抗菌药的敏感性也不断发生着变化。Ct抗菌药敏感性试验始于20世纪70年代,至今虽然已经发展了许多药敏检测方法,但是由于Ct生长周期长、培养要求高、影响因素多等特点,敏感试验多比较繁琐、耗时,至今尚无一种公认的标准方法可以用于测定Ct药物敏感性。目的:通过细胞培养法分离得到Ct临床株,并测定10种常用抗菌药在体外对Ct的抗菌能力,了解本地区性病门诊Ct药物敏感情况,并探讨Ct药敏的变迁及临床疗效与实验结果的关系。在Ct E型标准株的基础上,建立一套适应于我所实验室的成熟可行的Ct临床株培养筛查方法和药敏试验方法,并保留Ct临床株,为进一步深入研究奠定基础。通过药敏试验,为耐药机制的研究筛查耐药菌株。方法:收集了2005年4月-2007月3月期间到天津市性传播疾病研究所门诊就诊患者的尿道或宮颈拭子194例,所有患者均符合采集标准,并记录其临床相关资料。试验以Ct E型标准株培养为基础,采用McCoy对临床标本的进行培养,所有临床株进行二代盲传培养,对二代后阳性的标本继续传代培养。90%以上McCoy细胞形成包涵体的阳性标本扩大样本量并冻存于-80℃冰箱中备用。采用培养液倍比稀释法利用96孔板测定Ct的药物敏感性。首先使用Ct E型标准株探索药敏实验条件。先确定能引起90%以上细胞感染的Ct接种量;抗菌药的工作浓度参照文献,以文献推荐浓度为中间浓度,将10种常用抗菌药:红霉素、克拉霉素、阿奇霉素、四环素、多西环素、米诺环素、左氧氟沙星、司帕沙星、莫西沙星、利福平设六个等比稀释浓度。将在生长培养液中传代数次的McCoy细胞接种于96孔板37℃、5%CO₂条件下孵育18～24小时,形成致密单层的细胞,按接种量加入Ct菌液,离心、去上清、加入含不同浓度抗菌药的衣原体感染液,并设立阴性、阳性对照,同前条件孵育48小时后,经碘染色后镜下观察结果。镜下未见有Ct包涵体形成的最小稀释浓度为MIE即最低抑菌浓度。将试验结果与患者临床资料的相关性进行分析。结果:首先培养临床标本194例共得到52例阳性临床株,阳性率约为26.8%。其中90%以上的细胞形成包涵体的标本有23株。对这23株标本进行药敏测定显示:红霉素的MIC范围为0.125～2μg/ml;克拉霉素的MIC范围为0.004～0.064μg/ml;阿奇霉素的MIC范围为0.0313～1μg/ml;四环素的MIC范围为0.040～0.625μg/ml;多西环素的MIC范围为0.015625～0.125μg/ml;米诺环素的MIC范围为0.002～0.128μg/ml;左氧氟沙星的MIC范围为0.0625～0.5μg/ml:司帕沙星的MIC范围为0.016～0.128μg/ml;莫西沙星的MIC范围为0.015～0.24μg/ml;利福平的MIC范围为0.001875～0.03μg/ml.显微镜下观察发现随着药物浓度的增加,包涵体形态学发生改变,失去了正常的椭圆形,并且数量减少。试验筛查到8例红霉素的耐药菌株。结论:1、成功地摸索出临床株Ct培养的方法,并提高了阳性率;保留下临床菌株,为Ct的其它相关研究奠定基础。2。成功的建立了一套适合我实验室的Ct药敏研究;并筛查到8例红霉素耐药菌株证明了天津临床上已出现了对红霉素的耐药3.试验药敏结果和临床疗效存在较大差异。更多还原

【Abstract】 Chlamydia trachomatis is an obligate intracellular bacteria and can cause many diseases in human.Urogenital Ct infection, except NGU, it also causes many complications and serious psychoburden to the patient. According to the estimation of WHO, there are 93 million new cases every year and making severe economic burden to the society.Antimacrobials is the main drug for the treatment of Ct infection. It was thought that the microbiological cure rate of antimacrobials was above 90%, but the clinical treatment failure cases report more and more. There are many reasons associated with treat failure, factors like age of the patient and renewal of sexual intercouse, also the low compliance, drug pharmacolinetics and availability at the tissue level can dictate failue. And most of all the clinic drug resistant strains usually induce treatment failure; even forming persistence infection, resulting in infection unrecover for long term. As the antimacrobials’ alternation and rational use strengthened, the antimicrobial susceptibility of Ct changes unceasing.Objective: To investigate the in vitro effects of various antimacrobials against the clinic isolates of Ct, which are now mostly used in clinical. To understand the susceptibilitis of Ct in our clinical, moreover to explore the diversify of the susceptibilitis of Ct and the relation between the clinic curative effect and the experimental result. Establish a maturated feasible screening culture method and antimicrobial susceptibility testing method for clinical Ct which is suitable for our laboratory. Reserve the Ct positive clinical strains and it will lay a foundation for further research.Methods: Collecting 194 urethral or cervix swabs from the patients who came to Tianjin sexually transmitted disease institute between April 2005 to March , 2007. All of the patients were fit to the collection standard and we recorded their clinical related data. We undertook cell culture of clinical samples with McCoy cell on the foundation of Ct E standard strain. All the clinic strains were been blind passage cultured. The postive strain of the second generation are kept on culturing. When above 90% inclusions formed, we amplified this strain and reseved in refrigerator of 80 temperature below. First we explore the experiment conditions of the antimicrobial susceptibilities of Ct with Ct E standard strain. Then decide the inoculum size of the Ct, which can cause cell infection rate beyond 90%. Making the recommend concentration be the middle concentration, the ten antimacrobials(erythromycin、clarithromycin、azithromycin、tetracycline、doxycycline、minocycline、levofloxacin、sparfloxacin、moxifloxacin、rifamoin) are perpared for bexakis geometic proportion. We do the antimicrobial susceptibility test with 96-well plate. MIC was determined as the lowest concentration under which there were no inclusion forming, explore the relation between the clinic curative effect and the experimental result and screening the persister for the reseach of the drug resistance gene.Results: There were 52 culture positive clinical samples and 23sample infection rate beyond 90%. Minimal inhibitory concentration (MIC) of the 23clinical chlamydia trachomatis ranges were as follows: erythromycin,0.125～2 microgram /ml;clarithromycin,0.004～0.064microgram/ml;azithromycin,0.0313～1 microgra m /ml ; tetracycline , 0.040～0.625 microgram/ml ; doxycycline , 0.015625～0.125 microgram/ml;minocycline,0.002～0.128 microgram/ml; levofloxacin , 0.0625～0.5 microgram/ml ; sparfloxacin , 0.016～0.128 microgram/ml ; moxifloxacin , 0.015～0.24 microgram/ml; rifamoin, 0.001875～0.03 microgram/ml. Under the microscope, as the increasing of the drug concentration, the conclusions generate alteration in morphology , missing nomal oval and their quantity decreased. The results demonstrated that 8 wild strains is resistant to erythromycin.Conclusions: 1、Cultured Ct clinical samples from STD clinics in our department successfully and raised the positive rate; reserved the positive strains and lay foundations for further researches. 2、We grope a method suitable for clinical Ct strains cell culture and antimicrobial susceptibility test and found that eight wild isolate of Ct is resistant to erythromycin. In tianjin the clinical wild isolates of Ct have already been resistant to erythromycin . 4. we found that there are much difference between the clinic curative effect and the experimental result.更多还原