【作者】 左向华；

【导师】 陈建魁；

【作者基本信息】 中国人民解放军军事医学科学院 ， 免疫学， 2010， 硕士

【摘要】 目的在过去的几十年中,临床侵袭性曲霉感染(IA)的发病率一直持续上升,本病的病死率很高,可达50.0%～90.0%。而IA的早期诊断仍然是个急待解决的难题。对于免疫力低下的血液病和恶性肿瘤患者来说,及时的诊断和治疗IA是提高患者生存率和改善患者预后的关键。虽然组织病理学证据和活组织标本是确诊IA的金标准,但是这种侵入性的诊断方法在临床很难实施,尤其是免疫力低下及血小板减少的病人进行这种有创检查甚至会危及生命。国内外对于非侵入性的诊断方法一直在不断研究中,目前最有发展前景的是血清学方法和分子生物学方法。本课题研究了血清半乳甘露聚糖检测、血浆1,3-β-D葡聚糖检测以及联合检测在血液病和恶性肿瘤患者曲霉菌感染的早期诊断和抗真菌疗效中的价值;建立侵袭性曲霉菌病的Taqman Real-Time PCR诊断技术并对其在血液病和恶性肿瘤患者曲霉菌感染早期诊断中的价值进行初步探讨。方法GM和BG检测方法如下:每周两次同时采集临床具有真菌感染高危因素的226例血液病和恶性肿瘤患者的血清和血浆,分别采用ELISA Platelia Aspergillus试剂盒和MB-80微生物快速诊断系统检测GM和BG含量。根据根据2007年我国制定的血液病/恶性肿瘤患者侵袭性真菌感染(IFI)的诊断标准与治疗原则为标准,将研究对象分为:确诊IA和临床诊断IA(IA组)、拟诊IA组及排除IA组三个组。分别研究和对比两种诊断试验的评价指标。PCR检测方法如下:利用全自动核酸提取系统提取菌悬液和临床标本血清中DNA;利用Blast和DNASIS软件工具在GenBank数据库中查找曲霉菌属的高度保守序列。通过Primer Express2.0和Primer Express软件配合设计包括烟曲霉在内的五种曲霉菌通用引物和探针;设计扩增片段包括上述通用引物和探针的烟曲霉特异性引物,进行常规PCR扩增,扩增所得片段制备标准品并建立荧光PCR标准曲线;对临床血清标本中提取的曲霉菌DNA进行Real-Time PCR技术检测。结果GM与BG的检测结果:⑴血清GM检测对临床曲霉菌感染患者诊断的敏感性为84.4%,特异性为90.1%。以GM阳性为起点对临床高危患者及时抗曲霉治疗,病死率可减少至13.7%。⑵利用ROC曲线确立血浆BG检测在侵袭性真菌感染中的最佳阳性界值为20pg/ml,在此阳性界值下BG对侵袭性真菌的感染诊断的敏感性和特异性为88.3%和86.8%,对曲霉菌感染患者诊断的敏感性和特异性分别为88.9%和62.5%。曲霉菌感染患者和其他真菌患者的检测浓度相比较差异无统计学意义。⑶BG和GM联合检测对曲霉菌感染患者诊断的敏感性和特异性分别为82.2%和99.3%,与其单项检测相比较特异性得到很大提高。⑷GM和BG检测与临床其他检测方法相比较,27例患者GM检测阳性早于影像学诊断和痰培养3～10天,25例患者BG检测阳性早于影像学诊断和痰培养1～7天。⑸对以GM阳性为起点抗曲霉治疗的患者的GM和BG浓度进行监测,二者浓度变化相似,会随治疗有效而下降,也会随着治疗无效而增加。PCR检测方法结果:⑴菌液和血清中提取的DNA在普通PCR和Real-Time PCR分析都获得较好结果。⑵建立了Real-Time PCR检测曲霉菌DNA的技术平台,检测敏感性达到10copies/ml,用该方法对不同浓度的DNA模板进行扩增,在107-103Copies/ml时线性最好。⑶Real-Time PCR对临床曲霉菌感染患者诊断的敏感性为86.7%,特异性85.5%。结论根据上述检测结果,可以认为⑴血清GM检测对临床曲霉感染患者有较好的早期诊断价值。⑵以20pg/ml为阳性界值时,BG对IFI诊断有较好的诊断价值,单独检测BG对IA诊断特异性较低,对IA诊断仅有较好的提示作用,无法从血浆BG浓度高低上来区分曲霉菌和其他其他真菌感染。⑶B G和GM联合检测大大提高了对IA诊断的特异性,降低假阳性的发生率,提高临床诊断的准确性。⑷动态监测BG和GM的浓度变化,对临床治疗不仅有良好的指导意义,还有利于判断疗效和预后。⑸初步建立了曲霉菌感染的TaqMan Real-Time PCR诊断技术,该技术应用于临床对IA感染有较好的早期诊断价值。更多还原

【Abstract】 objectiveThe incidence of invasive aspergillosis(IA) has increased dramatically during the last decade. These infections are associated with high mortality, ranging from 50% to 90%, especially in hematological diseases and malignant tumor patients.The critical problem is the difficulty in making the early diagnosis. Unfortunately, delayed diagnosis and treatment for IA are associated with poor outcomes and high mortality regardless of therapeutic modalities used. Tissue biopsy or histopathological specimens which involves invasive procedures are still considered the gold standard for establishing the diagnosis. However, these procedures are rarely performed, especially in the setting of immunosuppression or patients with thrombocytopenia, where such invasive procedures can be life-threatening. There has been an increased search for better noninvasive diagnostic methods for IA. At present, the serum antigen detection and molecular detection is the focus of research。This subject studied the clinical value of (1,3)-β-D-glucan(BG) antigen detection、galactomannan(GM) antigen detection and combined detection in early diagnosis and anti-fungal effect in hematological diseases and malignant tumor patients. We expect to develop and evaluate a Taqman Real-Time fluorescence PCR diagnostic techniques for IA and preliminary study the clinical value of this method in early diagnosis of IA .MethodsGM and BG detection are as follows:The serum and plasma samples obtained from 226 patients were detected with platelia Aspergillus assay kit and MB-80 microorganism rapid detection system. According to the hematological disease/malignant tumor patients with invasive fungal infections (IFI) diagnostic criteria and treatment principles as the standard: The patients were divided into three groups,including diagnosis and clinical diagnosis of IA (IA group)、clinical suspected IA group and non-invasive Aspergillus infection group.PCR detection method is as follows: we extracted bacterial and serum DNA in clinical specimens by automatic nucleic acid extraction system; According to the GenBank database, we used the Blast and DNASIS tools to search highly conservative sequence of Aspergillus. One pair of Real-Time PCR universal primers and fluorescent probes of Aspergillus were designed by Primer Express 2.0 and Primer Express software; The standards were established and used to establish standard curves. The Real-Time PCR method were used to detected the Aspergillus DNA in clinical patients.ResultsGM and the BG test results:⑴A ccording to results of GM antigen detection,the sensitivity and specificity of serum GM test were 84.4% and 90.1%. The mortality can be reduced to 13.7% when the clinical anti-Aspergillus treatment were administrated according to positive results of GM.⑵Based on ROC curve,we can achieved the optimal results using the positive value of 20pg/ml for plasma BG detection in invasive fungal infections. The BG detection for diagnosis of IFI has shown sensitivity and specificity were 88.3% and 86.8%. The BG detection for diagnosis of IA has shown sensitivity and specificity were 88.9% and 62.5 %. The comparison of plasma BG concentrations between Aspergillus infections and other fungal infections had no statistical significance(P>0.05).⑶The combined detection of BG and GM for diagnosis of IA has shown sensitivity and specificity were 82.2% and 99.3%.⑷Compared with conventional detection methods,the positive results in 27 patients were obtained using GM detection method which preceded the findings on imaging diagnosis and sputum cultures about 3～10 days. 25 patients’BG detection preceded the findings on imaging diagnosis and sputum cultures about 1～7 days.⑸the concentrations of GM antigen and BG antigen decreased when anti-fungal treatment was effective for IA and increased as the treatment ineffective.Real-Time PCR detection results:⑴D NA extraction from fungal suspensions and serum can obtain a good result by real-time PCR and general PCR and analysis.⑵The technological platform for rapid detection of Aspergillus was established using Real-Time PCR. With this method,different concentrations of DNA templates were amplified,the best linearity range was 107-103Copies/ml; the sensitivity of detection is 10copies/ml,⑶Real-Time PCR for clinical diagnosis of IA in patients has shown sensitivity and specificity were 86.7% and 85.5 %.ConclusionAccording to the above results, we got five conclusions:⑴The detection of GM antigen has shown high specific and sensitive. The GM assay has a guiding value in the early diagnosis of IA .⑵The positive value of 20pg/ml of plasma BG is the most optimal for IFI early diagnosis. BG detection has a good clinical diagnosis value of the IFI. BG assay has low specificity for clinical diagnosis IA.⑶The combined detection of BG and GM can reduce false-positive rate ,improve the specificity and clinical diagnostic accuracy in the diagnosis of early IA.⑷GM and BG assay is a good indicator for antifungal therapy in IA patients . Monitoring of GM and BG concentration has been shown to be a a clinical index for appraising the therapeutic efficacy and prognosis of IA patients.⑸The Real-Time quantitative PCR techniques for IA was preliminary established. This method was useful for screening Aspergillus spp. in patients with high risk IFI factors and it has a good early diagnosis value of IA in hematologic diseases and malignant tumour patients.更多还原