【作者】 符美华；

【导师】 刘维达； 陈祥生； 吕桂霞； 崔盘根；

【作者基本信息】 中国协和医科大学 ， 皮肤病与性病学， 2005， 博士

【摘要】 近几十年来,随着科技的发展和医学的进步,广谱抗生素、糖皮质激素、免疫抑制剂的广泛应用,器官移植和各种导管技术的发展,免疫缺陷人群(AIDS患者,恶性肿瘤和血液病患者,吸毒者,等等)的扩大,机会性深部真菌感染的发病率明显增加。在免疫受损患者中,深部真菌的感染仍以念珠菌和曲霉最为常见。念珠菌属引起的感染占院内真菌性血行感染(BSIs)的四分之三,已成为美国院内血行感染的第四大病因。且由于该类患者的基础状况较差,如果不能早期诊断及合理治疗,死亡率较高。随着高危人群中预防性应用唑类药物的增多,系统性和侵袭性念珠菌感染的致病菌谱发生了相应变化,白念珠菌虽是念珠菌属发生机会性感染最主要的致病菌,但由非白念珠菌(NAC)引起的感染呈逐年上升的趋势,如光滑念珠菌、克柔念珠菌、近平滑念珠菌、季也蒙念珠菌和近年来新出现的葡萄牙念珠菌、挪威念珠菌、溶脂念珠菌和都柏林念珠菌等。光滑念珠菌和克柔念珠菌因其对唑类药物天然不敏感甚至耐药而受到越来越多的关注。及时准确地鉴定这两种念珠菌,对临床药物的合理选择具有重要意义。目前,深部念珠菌病的诊断主要依靠镜检和培养及组织病理,念珠菌的培养一般需要2-3天,且阳性的比例不高,而念珠菌的表型鉴定方法如糖同化试验、CHROMAgar、API-20系统等因耗时较长且人为因素干扰大而限制了其临床应用。这就要求临床寻找更快速、经济、方便和准确的诊断方法。本研究欲对模拟的临床念珠菌感染的标本进行真菌通用引物的盲法PCR检测,同时用白念珠菌、克柔念珠菌和光滑念珠菌的种特异性引物进行三重PCR检测并对试验程序和方法进行优化,评价分子诊断的特异性和敏感性,以期为临床可疑念珠菌病的早期诊断提供一个快速、方便和准确的方法。第一章:PCR实验方法的建立。首先验证真菌通用引物PCR检测真菌DNA的特异性。对临床常见的白念珠菌、光滑念珠菌、克柔念珠菌、热带念珠菌、烟曲霉、新生隐球菌用通用引物扩增出260bp左右的目的片段。而对临床常见的易被念珠菌感染的无菌体液,如血液、尿液、肺泡灌洗液和脑脊液等,应用真菌通用引物进行PCR扩增,未出现目的片段。其次,验证白念珠菌、克柔念珠菌、光滑念珠菌种特异性引物的特异性。对纯培养的上述三种真菌分别进行种特异性引物的PCR扩增,分别扩增出402bp、475bp、632bp左右的目的片段,而对热带念珠菌、烟曲霉、新生隐球菌、血液、尿液、肺泡灌洗液、脑脊液等,均未扩增出目的片段。第三,验证上述四种引物的敏感性。在生理盐水、血液、尿液、肺泡灌洗液和脑脊液中分别加入上述三种念珠菌,制成10~6-10~1CFU/ml的临床模拟标本,进行PCR扩增,并调整反应条件和反应体系,可检测出DNA的最低菌量为10~2CFU/ml,并多次重复实验,保证实验的稳定性和可重复性。第四,建立针对白念珠菌、克柔念珠菌、光滑念珠菌的三重PCR方法。对白念珠菌、克柔念珠菌、光滑念珠菌进行不同浓度的混合,用上述三种念珠菌的特异引物进行PCR扩增,并通过调节三种引物和ITS4的浓度,获得了较高的敏感性。最后,建立播散性念珠菌病的兔模型。用静脉接种白念珠菌的方法,成功建立播散性白念珠菌病的动物模型。第二章:对深部念珠菌病模拟标本进行三重PCR检测,评估PCR方法检测深部念珠菌病的稳定性、敏感性和特异性。首先,对临床常见的深部真菌感染的不同体液标本中,真菌通用引物和白念特异引物检测的敏感性进行比较;丝状真菌(烟曲霉)和酵母菌(白念珠菌)检测的敏感性进行比较;模拟体内环境,在EDTA抗凝血加入不同浓度的白念珠菌,37℃孵育,比较不同时间段PCR检测标本中真菌的敏感性。其次,设计随机表,请实验不相关人员配置不同浓度的白念珠菌、克柔念珠菌、光滑念珠菌菌悬液,按照随机表分别加入到血液、尿液、肺泡灌洗液和脑脊液中,制成不同念珠菌、不同浓度、不同体液的深部念珠菌病的模拟标本。对上述标本进行真菌培养,了解真菌培养的阳性率。同时对模拟标本进行真菌通用引物的单重PCR检测和3种念珠菌种特异性引物的三重PCR检测,以期通过一次PCR实验,能把白念珠菌、克柔念珠菌和光滑念珠菌彼此分开。同时评价PCR检测念珠菌感染模拟标本的稳定性、敏感性和特异性。设计随机表,请实验不相关人员按随机表在血液中加入不同浓度的白念珠菌、克柔念珠菌和光滑念珠菌,制成不同浓度混合的白念珠菌、克柔念珠菌和光滑念珠菌的模拟血液标本。对模拟标本首先在CHROMAgar显色培养基上培养。对相同的标本用真菌通用引物进行单重PCR检测和白念珠菌、克柔念珠菌和光滑念珠菌特异引物三重PCR检测,了解PCR检测混合模拟标本的稳定性、敏感性和特异性。结果显示,真菌培养和三重PCR检测深部念珠菌病临床模拟标本的阳性率的差异没有显著性,而三重PCR检测的时间较短,在6h内能完成一次实验,大大缩短了深部念珠菌病临床模拟标本的检出时间,可以用于深部念珠菌病的临床标本的检测。第三章:播散性白念珠菌感染兔模型的构建和药物疗效的分子监测。通过静脉内接种的方法,构建播散性白念珠菌感染的兔模型。在接种后24小时,用伊曲康唑注射液5mg/kg对兔模型进行治疗,每天一次,共14天。在不同的时间段取兔模型的静脉血,进行血培养和真菌通用引物和白念珠菌特异性引物的PCR检测,监测伊曲康唑注射液治疗播散性白念珠菌感染的疗效。同时对兔模型进行体温监测和外周血的白细胞监测,了解在真菌感染和治疗的过程中,兔模型的一般情况。结果:在接种白念珠菌后1h、6h,外周血中用PCR方法就能检测到白念珠菌,且能持续到8—10天;实验兔外周血血培养1h后阳性,持续到18h血培养阳性。伊曲康唑注射液治疗组较对照组体温下降较早。实验结束后解剖实验兔,治疗组较对照组内脏器官的组织培养阳性率及菌落数低。结果表明,PCR是一种快速和敏感的检测播散性念珠菌病的方法,伊曲康唑注射液治疗播散性白念珠菌病有效,但是真菌的清除率特别是肾脏组织的真菌清除率并不理想。小结:在本研究中,我们成功地建立了检测深部念珠菌病的PCR方法,并用盲法评价了该方法检测深部念珠菌病临床模拟标本的敏感性、特异性和稳定性。成功建立了播散性白念珠菌病的兔模型,用分子方法评价了伊曲康唑注射液治疗播散性白念珠菌病的药物疗效。更多还原

【Abstract】 The progress of medical sciences and universal application of new treatments in recent years such as broad-spectrum antibiotics,systemic use of corticoids, antimalignancy chemotherapy and organ transplantation,have resulted in a sharp increase in the number of immunocompromised patients.Immunosuppression is occasionally accompanied with fungal infections of an insidious onset,and some nosocomial.Invasive mycoses have become a major cause of infectious morbidity and mortality in patients receiving immunosuppressive chemotherapy for cancer or organ transplantation.On the other hand,invasive mycoses are not uncommon in immunocompromised patients,such as individuals with AIDS,cancer,drug abuse. More than 150 species of fungi are known to be responsible for infections in human. All of tissues and organs can be involved.Since opportunistic mycoses are often clinically grave and serious,an early,rapid and accurate identification of the pathogenic fungus is critical to timely and appropriate management.Due to growing application of azoles to prevent mycoses,the fungal infections by non-albicana candida (NAC)has seen a steady rise.C.krusei and C.glabrata are the two most common causes of NAC infections.Nonetheless,C.albicans is the most important in invasive cases.Since C.krusei and C.glabrata are naturally resistant to azoles,they are gaining more and more attention.Fungi are traditionally identified by morphology and metabolic characteristics and they reqiure days even weeks to be isolated on cultures. Identification may sometimes be difficult,which would impede the selection of antimicrobial agents in cases in which species identification of the pathogen is vital. Therefore,we devised a rapid and accurate method of identification,especially to identify C.albicans,C.krusei and C.glabrata.In this study,we developed a universal fungal primer for panfungal PCR and multiplex primers for triplex PCR of C.albicans, C.krusei and C.glabrata.Chapter 1 Development of universal fungal primer PCR and specific primer PCR for detection of common deep-seated pathogenic fungi.First,in order to test the specificity of the PCR assay,the universal fungal primer was used to amplify nucleinic acid of C.albicans,C.krusei,C.glabrata,C.tropicalis,Aspergillus fumigatus,Cryptococcus neogenesis,a 260bp PCR product was successfully obtained from these fungi,while human blood,urine,bronchoalveolar lavage,cerebrospinal fluid samples produced negative results.Second,a 402bp PCR product was successfully amplified from C.albicans,a 475bp one from C.krusei,a 632bp one from C.glabrata,while C.tropicalis,Aspergillus fumigatus,Cryptococcus neogenesis,human blood,urine,bronchoalveolar lavage,cerebrospinal fluid samples were negative.Third,serial dilutions of fungal DNA of C.albicans,C.krusei, C.glabrata were amplified with panfungal primer to detect the sensitivity of the PCR. The sensitivity of PCR was 10~2 CFU/ml.The detection of fungal DNA in simulated clinical specimens with the panfungal PCR was explored with human blood,urine, bronchoalveolar lavage,cerebrospinal fluid.Fourth,we developed multiplex primers for C.albicans,C.krusei and C.glabrata for the triplex PCR.The products of 402bp,475bp and 632bp were successfully amplified from the mixture of C.albicans, C.krusei and C.glabrata and the simulated clinical specimens.Finally,rabbit models of disseminated C.albicans infection were constructed through injecting C.albicans intravenous inoculation.Chapter 2 Detection of the simulated clinical specimens with the triplex PCR. First,we compared the sensitivity of PCR in different fluids and the sensitivity of PCR to C.albicans and Aspergillus fumigatus.The blood simulated specimens of C.albicans were cultured at 37℃.The sensitivity of the specimens were deteceted at the different points of time during the course.Second,according to the random table, we added concentrationnally different suspensions of C.albicans,C.krusei and C.glabrata into human blood,urine,bronchoalveolar lavage,cerebrospinal fluid samples.We detected the simulated clinical specimens with the triplex PCR.Simultaneously we cultured the same specimens on SDA medium.The results showed that the positive rate of triplex PCR was not statistically different from that of fungal culture with respect to detecting deep-seated Candidosis,but the time of triplex PCR requied was much shorter than that of fungal culture.The PCR can be acoomplished in 6 hours.As a result,triplex PCR can serve to efficiently detect the clinical specimens of the deep-seated candidosis.Chapter 3 Construction of rabbit models of disseminated C.albieans infection.Rabbits were challenged with C.albicans through intravenous inoculation.The disseminated infection occurred within 1 hour and 6 hour after challenging.At 24 hour,the rabbits begun to be treated with itraconazole injection 5mg/kg,qd×14d.At every point of time,we collected the venous blood of the rabbits and detected the blood with universal fungal primer and specific primer of C.albicans to evaluate the effect of itraconazole treatment.At the same time,the specimens were cultured on SDA medium.The body temperature and white blood cell count of the rabbits were monitored simultaneously.The fungal culture were positive within 1hour after challenging and continued up to 18 hours.After the experiment,the rabbits were dissected.The positive rate and CFU of organs and tissues culture were higher in treated group than the control.The conclusions can be made that PCR is rapid and sensitive in detecing disseminated candidosis and itraconazole injection is effective in treating the disseminated candidosis,but the clearance rate were not so satisfactory esspecially for the fungi in kidney.Conclusion:In this blinded study,we successfully developed the PCR to detect the deep-seated candidosis and evaluated the sensitivity,specificity and reliability of the PCR on detecting the simulated specimens of deep-seated candidosis.The PCR is highly efficient in detecting deep-seated candidosis.The rabbit models of disseminated candidosis were successfully constructured and the effecacy of itraconazole injection in treating the disseminated candidosis were evaluated by molecular method.更多还原