【作者】 李梦；

【导师】 廖万清；

【作者基本信息】 第二军医大学 ， 皮肤病与性病学（专业学位）， 2013， 博士

【摘要】 选题背景：近年来，随着分子生物学技术的迅猛发展，多种分子生物学技术如P C R指纹分型，扩增性片段长度多态性，核糖体r D NA序列分析，多位点序列分型等相继用于隐球菌的分子流行病学及遗传多态性研究，极大地促进了人类对新生隐球菌遗传多态性的认识，增强了寻找致病线索的信心。通过分子生物学基因分型的方法了解各地流行的隐球菌基因型，对明确隐球菌的发病机制及流行趋势有着深远的意义。采用适当的分子生物学分析方法对临床隐球菌感染患者体液中分离出的菌株进行基因分型，有助于了解我国临床隐球菌的分子流行病学特征并监测各基因型的变迁情况。结合隐球菌临床分离株对常见抗真菌药物的敏感性和临床患者的临床表现、治疗效果及预后，以揭示基因型、体外药敏实验和临床治疗之间的关联。本课题分析了61株隐球菌临床分离株的基因型和对两性霉素B、氟康唑、伊曲康唑、氟胞嘧啶的体外药物敏感性，分析了基因型和体外药敏的关联。另外，如何更早更准确的明确隐球菌病的诊断对于临床早期开展抗真菌治疗和改善临床预后至今仍是目前临床诊疗亟需解决的众多问题之一，本课题还尝试结合隐球菌特异性基因和目前已有的分子技术方法中，寻求新的更快速的诊断方法。研究目的：1.分析1993-2009年分离自我国临床感染患者的61株隐球菌的基因型组成2.观察61株隐球菌临床株对两性霉素B、伊曲康唑、氟康唑、氟胞嘧啶的体外药物敏感性；3.分析不同种属、不同基因型的隐球菌对药物敏感性的差异；4.基于隐球菌H99的IGS1基因序列，结合LAMP技术，快速特异的诊断隐球菌感染。研究方法：1多位点基因序列分析及基因分型多位点基因序列分析：对61株隐球菌临床株的IGS1和CAP59基因进行PCR扩增、测序、序列分析；将各隐球菌临床株鉴定至基因亚型。2.观察隐球菌临床株对常见抗真菌药物的敏感性用美国CLSI的M27A2微量稀释法对61株隐球菌临床株进行常见抗真菌药物两性霉素B、伊曲康唑、氟康唑、氟胞嘧啶的药敏实验。3.比较不同基因型的隐球菌临床株对常见抗真菌药物敏感性的差异4，构建新的快速、特异、敏感的方法以检测隐球菌基于新生隐球菌H99的IGS1序列，利用LAMP(环等温介导扩增)方法，通过检测隐球菌不同基因型的菌株和其他常见真菌验证其特异性，根据可检测出的最小DNA浓度判断其敏感性。结果：1.多位点基因序列分析及基因分型分离自临床感染患者的61株隐球菌临床株中，45株（73.77%）为VNI基因型菌株；5株（8.20％）为VN II基因型菌株；2株（3.28％）为VN III基因型菌株；；14株（14.75％）为VGI基因型。IGS1基因结合CAP59基因序列分析可以明确的将各临床株鉴定至基因型水平。2.观察隐球菌临床株对常见抗真菌药物的敏感性61株隐球菌临床分离株对两性霉素B、氟康唑、伊曲康唑、氟胞嘧啶均敏感，最小抑菌浓度范围分别为0.0625~1μg/ml，0.313-4μg/ml,0.125~16μg/ml0.125~8μg/ml,MIC90分别为1μg/ml，0.25μg/ml，4μg/ml，4μg/ml。3.不同基因型的隐球菌临床株对常见抗真菌药物敏感性的差异（1）47株从非HIV感染患者体液中分离得到的菌株对氟康唑的MIC浓度为0.125-4μg/Ml，其几何平均数为1.1589μg/Ml；而14株来自HIV感染患者的临床分离株对氟康唑MIC浓度为0.5-16μg/Ml，其几何平均值为3.1228μg/Ml；其差异具有统计学差异（P=0.001）。（2）47株从非HIV感染患者体液中分离得到的菌株对氟胞嘧啶的MIC浓度为0.125-4μg/Ml，其几何平均数为1.4038μg/Ml；而14株来自HIV感染患者的临床分离株对氟康唑MIC浓度为0.5-8μg/Ml，其几何平均值为2.9720μg/Ml；其差异具有统计学差异（P<0.001）。（3）新生隐球菌对两性霉素B，伊曲康唑、氟康唑、氟胞嘧啶的MIC浓度分别为0.0625-1μg/Ml，0.313-4μg/Ml,0.125-16μg/Ml.0.125-8μg/Ml。格特隐球菌对这四种抗真菌药物MIC浓度为0.125-1μg/Ml,0.0313-0.5μg/Ml，0.5-4μg/Ml,0.5-4μg/Ml。从种间差异分析，新生隐球菌和格特隐球菌对于这四种抗真菌药物的差异不具有统计学差异。（4）从基因型分类角度，基因型VNⅠ对两性霉素B、伊曲康唑、氟康唑、氟胞嘧啶MIC几何平均值分别为0.5156μg/Ml,0.1122μg/Ml,1.3608μg/Ml,1.6371μg/Ml；而基因型VGⅠ则分别为0.6300μg/Ml,0.1072μg/Ml,1.8517μg/Ml,1.8517μg/Ml。这两种基因型的隐球菌对两性霉素B、伊曲康唑、氟康唑、氟胞嘧啶的MIC均无统计学差异。由于在基因型VNⅠ的菌株中有部分菌株来自于HIV感染阳性的患者，为了尽可能减少干扰因素，我们将基因型VNⅠ菌株中来自于HIV感染患者的菌株去除后，再次与基因型VGⅠ菌株分别就两性霉素B、伊曲康唑、氟康唑、氟胞嘧啶再次做Student’s比较，结果依然无统计学差异。4，构建新的快速、特异、敏感的方法以检测隐球菌基于IGS1序列设计的引物在LAMP反应体系具有良好的特异性，除了新生隐球菌和格特隐球菌八个基因型出现阳性结果外，其他菌株包括劳伦特隐球菌、浅白隐球菌、浅黄隐球菌、土生隐球菌、指甲隐球菌、瘦弱隐球菌、斯金纳隐球菌、念珠菌属中的白念珠菌、克柔念珠菌、热带念珠菌、近平滑念珠菌、都柏林念珠菌、毛孢子菌等均未出现扩增反应。基于新生隐球菌H99的核糖体内基因间隔区IGS序列的LAMP敏感程度可以达到0.002fg（即10-6ng）。结论：1.分离自我国临床患者的隐球菌临床株以VN I基因型菌株为主；分离自AIDS患者的临床株亦以VN I基因型菌株为主，2.我国的隐球菌临床株绝大部分对常见抗真菌药两性霉素B、氟康唑、伊曲康唑、氟胞嘧啶均敏感，存在个别菌株对两性霉素B的敏感性下降，可能与临床上治疗接触该药有关系，具体机制尚待进一步研究探讨；3隐球菌各基因型对这四种抗真菌药物的体外药敏无显著差异；4，来源于HIV阴性患者的临床株比来源HIV阳性的菌株对氟康唑和氟胞嘧啶更为敏感；5基于IGS1序列设计的引物在LAMP反应体系具有良好的特异性和敏感性，能够快速、准确、简便的鉴定新生隐球菌和格特隐球菌，可以进一步研究将该法应用于临床的隐球菌感染早期诊断技术之一。更多还原

【Abstract】 BackgroundRecently, the development of molecular techniques has greatly improved ourunderstanding of genetic diversity of Cryptococcus species complex。Several differentmolecular typing methods have been used in epidemiological and genotypic analysis of theCryptococcus species complex, including random amplified polymorphic DNA (RAPD),PCR fingerprinting, restriction fragment length polymorphism (RFLP) analysis, AFLPanalysis，sequencing analysis of single locus and multi locus.In this study, we determined the genotype of61Cryptococcus neoformans andCryptococcus gattii Complex strains isolate from HIV-positive and HIV-negative patientsby using the IGS1gene and CAP59gene sequencing analysis. The study of correlation ofspecies,genotype and in vitro susceptibility, the clinical outcome will plays an importantrole to guide clinical therapy. In the study,Microdilution method M27-A2program wasused to test the antifugal susceptibilities of clinical isolates t amphotericinB,fluconazole,itraconazole and flucytosine and we try to find the relationship betweengenotype and MICs.Otherwise, how to diagnosis the cryptococcal infection timely and excisely ismeaningful to initiating antifungal therapy and improving clinical outcome. We built a newmolecular method based on IGS1gene to detect Cryptococus.spp more quickly by usingLAMP.Objects1. multi locus sequencing typing and genotype determination2To investigate the antifugal susceptibilities of clinical isolates to amphotericin B,fluconazole, itraconazole and flucytosine3. To analysis the correlation of genotype and MICs4, To build a new molecular method to detect Cryptoccus. Spp by using LAMP Methods1. multi locus sequencing typing and genotype determinationA total of61Chinese clinical Cryptococcus neoformans species complex isolateswere analysed by sequences of IGS1gene and Capsule59gene, all of the strains weredetermined their genotype.2To investigate the antifugal susceptibilities of clinical isolates to amphotericin B,fluconazole, itraconazole and flucytosineMicrodilution method M27-A2program was used to test the antifugal susceptibilitiesof clinical isolates t amphotericin B,fluconazole,itraconazole and flucytosine3. To analysis the correlation of genotype and MICsAnalysis of susceptibility data with origins, species and genotypes.4, To build a new molecular method to detect Cryptoccus. Spp by using LAMPBuild a new molecular method based on IGS1gene combining the LAMP technique.To examine the specificity of the new method by detecing the Cryptococcal spp and other48fungal species. Sensitivity was tested by10-fold DNA dilution series.Results1. multi locus sequencing typing and genotype determinationIn all the61strains isolated from Chinese HIV-Negative and-Positive patients,. VNI(73.77%) was the most represented genotype,5strains(8,20%) were VNII genotype,2strains(3,28%) were VNIII genotype, and the rest (14,75%)were VGI genotype2To investigate the antifugal susceptibilities of clinical isolates to amphotericin B,fluconazole, itraconazole and flucytosineThe MIC90and susceptibility ranges for total Cryptococcus species complex isolateswere as follows:1(0.0625~1) μg/ml for amphotericin B,4(0.125~16) μg/ml forfuconazole,0.25(0.0313~4) μg/ml for itraconazole, and4(0.125~8) μg/ml forflucytosine.3. To analysis the correlation of genotype and MICs(1)When the isolates were analyzed according to the origin of the patients the isolatesfrom HIV-negative patients showed lower geometric mean for fluconazole (1.1589/3.1228, p=0.001) and flucytosine (1.4038/2.9720, p<0.001), compared with those from AIDSpatients.(2)There were no significant differences in susceptibilities between the species for eachagent(3)Although the geometric mean MICs were different, it showed no statistically significantdifference (P>0.05) between genotype VNI and VGI4, To build a new molecular method to detect Cryptoccus. Spp by using LAMPThe experiment using LAMP primers for Cryptococcus neoformans IGS1gene did notshow cross-reactivity with other fungi, the specificity assay showed that positiveamplification is only obtained with the genus Crypotcoccus neoformans and Cryptococcusgattii complex. The detection limit was found to be0.002fg.Cnclusion1. The isolates of VNI genotype is the most majority of clinical Cryptococcusneoformans species complex isolates from Chinese HIV-Positive and HIV-Negativepatients。2，Most of the clinical Cryptococcus species showed excellent suscepibitily to theamphotericin B, fluconazole, itraconazole and flucytosine. Whereas there was a high rateof tolerance against amphotericin B (MICs range from0.5-1μg/ml) in these clinicalisolates. These data suggested that amphotericin B as first-line therapy for Cryptococcusmay lead to the tolerance or even resistance of pathogen over time although the explicitmechanism is not clear so far.3,As for fluconazole and flucytosine, Cryptococcus neoformans isolates from AIDSpatients were less susceptible than those from Non-AIDS patients.4,The study of correlations of species, genotypes and in vitro susceptibilities showed nosignificant clinical meaning.5,The LAMP technique based on Cryptococcus neoformans H99IGS1gene enablesspecific deciton of Cryptococcus neoformans and Cryptococcus gattii Complex. Themethod provides a powerful tool for rapid diagnosis in the clinical lab.更多还原