【作者】 张宇一；

【导师】 吴菊芳；

【作者基本信息】 复旦大学 ， 内科学， 2011， 硕士

【摘要】 第一部分中枢神经系统感染病原快速基因诊断研究目的：评价病原快速基因检测方法在中枢神经系统感染中的可靠性与实用性,明确其临床应用价值。方法：按入选排除标准收集神经外科术后拟诊为中枢神经系统感染患者的脑脊液,通过PCR扩增及基因芯片杂交技术检测病原菌种类,并与常规脑脊液培养的结果进行比较。结果：共收集脑脊液标本204例,其中常规方法培养阳性者29例,基因芯片方法检测阳性者为25例。基因诊断方法与常规方法比较,符合率98.03%(200/204)敏感度为86.21%,特异度为100%。如排除因基因芯片未设置探针而检测阴性的3例标本,则符合率为99.50%(200/201),敏感度为96.15%。基因诊断较常规培养方法在病原菌的诊断时间上缩短3.24±1.22天；如就脑脊液细菌阳性病例而言,检出时间缩短2.44±0.68天；二者均有显著差异(P<0.01)。结论：病原快速基因检测方法与常规培养方法比较,符合率高、检测时间明显缩短,方法可靠,具有临床应用价值。第二部分人血浆及脑脊液中美罗培南浓度的HPLC测定方法研究目的：建立人血浆及脑脊液样品中美罗培南的定量测定方法。方法：采用高效液相色谱法,TSK-gel ODS-80TM C,8柱(150mm×4.6mm,5μm),血浆样品测定流动相为15mmol·L-1KH2P04(pH3.2)—乙腈(87：13),脑脊液样品测定流动相为lOmmol·L-1乙酸胺(PH3.86)—乙腈(90：10),紫外检测波长均为308nm。结果：血浆样品中美罗培南保留时间为3.148分钟,在0.5mg·L-1～100mg·L血药浓度范围内线性关系良好(r=0.9999),最低检测浓度为0.5mg·L-1；各质控浓度提取回收率平均为109.1%,日内、日间RSD均<10%,方法回收率为82.6%～96.6%；-80℃冰箱保存6月内回收率稳定。脑脊液样品中美罗培南保留时间为3.810分钟,在0.25mg·L-1～50mg·L-1脑脊液药浓度范围内线性关系良好(r=0.9989),最低检测浓度为0.25 mg·L-1；各质控浓度提取回收率平均为96.8%,日内、日间RSD均<5%,方法回收率为94.3%～106.3%；-80℃冰箱保存6月内回收率稳定。血浆及脑脊液样品检测时均使用PABA(对氨基苯甲酸)为内标物。结论：本研究建立的美罗培南检测方法灵敏度高、特异性强,适用于临床药代动力学的研究。第三部分美罗培南在中枢神经系统感染中的PK/PD研究目的：通过对术后中枢神经系统感染应用美罗培南患者进行血及脑脊液中美罗培南PK/PD研究,有望制定美罗培南治疗中枢神经系统感染的最佳治疗方案。方法：根据入选排除标准选择神经外科术后使用美罗培南的患者,并收集应用美罗培南后不同时间点的血、脑脊液标本及临床资料,测定药物浓度后计算PK/PD数据,以确定最佳给药方案。结果：美罗培南2g q8h给药方案的%T>MIC、Cmax/MIC及血脑屏障穿透率明显高于其余二组给药方案。对于鲍曼不动杆菌,当MIC值小于0.5mg/L时,三个给药方案在给药间隔内血和脑脊液药物浓度均高于MIC,%T>MIC为100%;MIC小于2mg/L时,血及脑脊液%T>MIC均大于50%左右；当MIC大于2mg/L时,脑脊液中该值不理想。当MIC值小于4mg/L时,血浆Cmax>MIC在5倍以上；与血药浓度相比,脑脊液中该值在MIC大于0.25mg/L时仅有2g q8h给药方案可达5倍的比值。结论：美罗培南治疗中枢神经系统感染2g q8h的给药方案为最优,其次为1g q6h,1g q8h,在治疗同时去除脑室或脑脊液外引流装置有助于病原菌的清除。更多还原

【Abstract】 Part 1 Study on the etiologic genetic diagnosis of central nervous system infectionsOBJECT:To know the clinical value of genetic method in central nervous system infections.METHODS:The cerebrospinal fluid of patients, who were suspected of central nervous system infection, was collected. The consistency rate between genetic method and traditional method were compared.RESULTS:204 cases of cerebrospinal fluid samples were collected,29 were positive as assessed by culture and 25 were positive as assessed by PCR. Compared with the traditional method, genetic method had a sensitivity of 86.21% and specificity of 100%. If removed 3 samples whose microarray probes were not set, the compliance rate was raised to 99.50%, and the sensitivity was 96.15%. Genetic method detection can help us get the result 3.24±1.22 days before the traditional method detection (P<0.01)CONCLUSION:We can draw the conclusion that genetic diagnostic method had good consistency with traditional method, and detection time was shortened. The new method is reliable and has clinical application value.Part 2 Determination of meropenem in human cerebrospinal fluid and plasma by HPLCOBJECT:To establish a HPLC method for determination of meropenem in human cerebrospinal fluid.METHODS:Chromatorgraphic separation was performed on a TSK-gel 0DS-80TM C18 column. Mobile phase of CSF was ammonium acetate and acetonetrile (90:10, v/v), Mobile phase of Plasma was KH2PO4 and acetonetrile (87:13, v/v). Ultraviolet absorption at 308nm was used for detection. The temperature of column was 35℃.After protein was precipitated by acetonetrile,the derivatized sample was eluted and 20μL injected. Para-aminobenzoic acid (PABA) was used as an internal standard.RESULTS In human cerebrospinal fluid, the retention time of meropenem was 3.810min and the detector response was linear over the concentration range of 0.25-50mg·L-1. The recovery was 94.3-106.3%, and relate standard deviation (RSD) of intra-day and inter-day assays were all less than 5%. In human plasma, the retention time of meropenem was 3.148min and the detector response was linear over the concentration range of 0.5-100mg·L-(?)The recovery was 82.6-96.6%, and relate standard deviation (RSD) of intra-day and inter-day assays were all less than 10%. CSF and plasma stored at-80℃for six months at various concentrations in cerebrospinal fluid, meropenem did not reveal any appreciable degradation.CONCLUSION The method is simple, sensitive and accurate, and can be used for the pharmacokinetic study.Part 3 PK/PD of meropenem in central nervous system infectionsOBJECT:To explore the PK/PD of meropenem in Central Nervous System Infections.Thus we can optimize the treatment of meropenem.METHODS:Neurosurgical patients, who were treated by meropenem, were selected based on inclusion criteria and exclusion. We collected blood and CSF samples on schedule, and got drug concentration by HPLC. At the same time, we collected their clinical data. Then we calculated PK/PD data, and determined the optimal dosage regimen.RESULTS:The dosage group which was received 2g(q8h) of meropenem has a maximum value of %T>MIC and Cmax/MIC, and also has the best blood-brain barrier penetration. For Acinetobacter baumannii, when MIC<0.5mg/L, the plasma concentrations and CSF concentration of three groups were higher than MIC in the dosing interval (%T>MIC was 100%).When MIC<2mg/L, the value of %T>MIC(both in the plasma and csf)was more than 50%. If MIC2mg/L, the value of %T>MIC (csf) was not ideal. When the MIC value is less than 4mg/L, the Cmax>MIC (plasma) in more than 5 times. But only in 2g q8h dosage group, the Cmax>MIC(CSF) is 5 times when MIC.25mg/L.CONCLUSION:To cure Central Nervous System Gram-negative Bacterial Infections,2g q8h dosage regimen is the best, followed by 1g q6h, 1g q8h. removing the drainage system would be useful of pathogen removal.更多还原