分子分型在医院感染控制中的作用课件

Click to edit Master title style,Click to edit Master text styles,Second level,Third level,Fourth level,Fifth level,.,*,分子分型在医院感染控制中的作用,（,the Role of Molecular Typing in Nosocomial Infection Control,）,瑞金医院临床微生物科,瑞金医院医院感染办公室,杨 莉,1,.,分子分型在医院感染控制中的作用（the Role of M,分子分型（分子流行病学研究）,从核酸分子水平上分析医院感染的发生、发展规律及机理，更加准确有效地进行医院感染管理控制，已成为当前国际医院感染管理研究中的重要方向。,从患者分离株到病区周围环境株的比较分析；,从外源性感染到内源性感染,从某一医院的医院感染暴发到大范围甚至世界范围的感染菌株流行变迁；,基因多态性分析技术已成为医院感染监测控制的,高水平研究领域。,2,.,分子分型（分子流行病学研究）从核酸分子水平上分析医院感染的发,微生物室在医院感染中的作用,accurately identifying nosocomial pathogens,detecting unexpected antimicrobial-drug resistance,epidemiologic typing,Pfaller MA et al. The clinical microbiology laboratory and infection control: emerging pathogens, antimicrobial resistance, and new technology. Clin Infect Dis 1997;25:858-70.,鉴定,特殊耐药菌的检出,流行病学分型,3,.,微生物室在医院感染中的作用accurately identi,PFGE(,脉冲场凝胶电泳),RAPD(,随机扩增,DNA,多态性),REA(,限制性酶切),ribotyping (,核糖体分型),分子分型,基因分型,分子流行病学研究,4,.,PFGE(脉冲场凝胶电泳)分子分型4.,1、脉冲场凝胶电泳(PFGE),制备琼脂糖栓块,消 化,酶 切,脉冲电泳,结果解释,5,.,1、脉冲场凝胶电泳(PFGE)制备琼脂糖栓块消 化酶,消化、释放出,DNA,PFGE,原理,6,.,消化、释放出DNAPFGE原理6.,Enzyme,酶切,PFGE,原理,约520个、长度10800,kb,的大片段,DNA,7,.,Enzyme酶切PFGE原理约520个、长度10800k,Tenover FC.et al.J Clin Microbiol,1995;33(9):22332239,8,.,Tenover FC.et al.J Clin Microb,脉冲电泳,PFGE,原理,9,.,脉冲电泳PFGE原理9.,原理：,DNA,常规凝胶电泳,脉冲场凝胶电泳,10,.,原理：DNA常规凝胶电泳脉冲场凝胶电泳10.,时间消耗,从分离菌株到出结果平均 2.5 天,标本准备、细胞裂解第一天,酶切第二天,染色、拍照第三天,PFGE,原理,染色、拍照,11,.,时间消耗PFGE原理染色、拍照11.,12,.,12.,PFGE,结果判读,目测法：,按美国疾病控制和预防中心(,CDC)Tenover,等人推荐的方法判读。,图谱完全相同的定为,一个型,，彼此之间相差一个带的定为同一型的,不同亚型,，相差23个带的认为,亲缘关系密切,，相差46个带的认为,可能相关,，条带相差7个以上的认为,无亲缘关系,。并随机地选择不同的字母如,A、B、C、D,等的字母顺序分型。,聚类分析,计算机输入,SPSS，,做树状图,13,.,PFGE结果判读目测法：13.,PFGE同源性分析,14,.,PFGE同源性分析14.,PFGE,特点：,DNA,原位提取法，减少了断裂,利用细菌全基因组信息,细菌分型金标准,15,.,PFGE特点：DNA原位提取法，减少了断裂15.,2、RAPD（随机扩增DNA多态性）,1990年,Williams：RAPD,Welsh：AP-PCR,本质上相同,引物：,“短” 、“单一” 和“非特异性”，一般910,bp,扩增条件：,“非严格性”,退火温度一般较低,2535,16,.,2、RAPD（随机扩增DNA多态性）1990年引物：16.,ESHWAR MAHENTHIRALINGAM,.et al.J Clin Microbiol,1996;34(5):11291135,17,.,ESHWAR MAHENTHIRALINGAM.et al.,ESHWAR MAHENTHIRALINGAM,.et al.J Clin Microbiol,1996;34(5):11291135,18,.,ESHWAR MAHENTHIRALINGAM.et al.,ESHWAR MAHENTHIRALINGAM,.et al.J Clin Microbiol,1996;34(5):11291135,19,.,ESHWAR MAHENTHIRALINGAM.et al.,分子分型在医院感染控制中的作用,20,.,分子分型在医院感染控制中的作用20.,Special Issue,New Technology for Detecting Multidrug-Resistant Pathogens in the Clinical Microbiology Laboratory,Lance R. Peterson* and Gary A. Noskin*Northwestern Memorial Hospital and Northwestern University Medical School, Chicago, Illinois, USA,EID, 2001,7: 306,21,.,Special IssueEID, 2001, 21.,Northwestern Memorial Hospital,Chicago,700-bed, university-affiliated medical center,出院：,39,000,/年,急诊：56,000例/年,门诊量：260,000/年,22,.,Northwestern Memorial Hospital,分型确认后及干预效果,P,=0.002,LR Peterson,et al, EID, 2001, 7: 306,5.79,23,.,分型确认后及干预效果P=0.002LR Peterson5.,指标,the total number of nosocomial infections per 1,000 patient days,每千住院日医院感染数,the number of patients with nosocomial infections per 100 patient discharges,每100出院病人医院感染病人数,(percentage of patients with nosocomial infection),（,医院感染病人百分比）,24,.,指标the total number of nosocomi,感染控制工作小组成员：,We formed a permanent, integrated infection control and prevention program that fully incorporates,Hacek DM et al. Am J Clin Pathol 1999;111:647-54.,infection control personnel,infectious disease personnel,pharmacy personnel,clinical microbiology personnel,into a single working group to minimize hospital infections.,感染控制,感染性疾病,药学,临床微生物,25,.,感染控制工作小组成员：We formed a permane,资料收集方法（,Methods for data collection,）：,review of microbiology reports,review of patients medical records,direct observation of medical and nursing practice,active surveillance of rectal cultures of patients in nursing units for high-risk patients,evaluation of suspected nosocomial infections reported by health-care providers.,查阅微生物报告,查阅病史记录,对医护人员操作的直接观察,高危病人直肠培养的动态连续观察,对上报的可疑医院感染的评估鉴定,由三位专职感染控制人员对资料进行汇总分析，并制定出相应控制措施，并在医院感染控制和预防部门主管的指导下实施。,26,.,资料收集方法（Methods for data collec,Two interventions：,a molecular typing laboratory,a weekly planning meeting,infection control,diagnostic medical microbiology (molecular epidemiology),Pharmacy,and infectious diseases,两个主要的干预措施：,分子分型,周会,包括以下方面的代表：,感染控制,微生物诊断（分子流行病学）,药学,感染性疾病,27,.,Two interventions：两个主要的干预措施：27,周会内容：,医院感染动向（短期、长期）,感染控制专职人员和微生物实验室的工作,决定需要做的调整,需分型的病原体与主管讨论决定,weekly meetings：,the ongoing short- and long-term trends in nosocomial infections within the center,activities of the infection control professionals and microbiology laboratory personnel;,any needed changes were determined.,The organizational structure for selecting microbes for typing was shared by the medical directors of infection control and clinical microbiology,28,.,周会内容：weekly meetings： 28.,需基因分型的微生物：,routinely genomically typed：VRE,Periodic routine typing：,fluoroquinolone-resistant,P. aeruginosa,methicillin-resistant,Staphylococcus aureus,(MRSA),Enterobacter cloacae,Clostridium difficile,Additional organisms for typing：selected through surveillance of microbiology culture reports discussed at the weekly meeting.,常规基因分型：,VRE,周期性分型：,氟喹诺酮耐药的铜绿,MRSA,阴沟肠杆菌,难辨梭菌,其他：根据微生物报告并在周会上讨论后决定需基因分型的病原体,任何时候,工作小组要求进行基因分型，临床微生物实验室即将菌株提供给,分子分型部门,并执行,29,.,需基因分型的微生物：routinely genomicall,实验方法,REA analysis,restriction of genomic DNA with conventional electrophoresis,DNA,限制性酶切,30,.,实验方法REA analysis30.,基因分型相关费用,the cost of equipment, remodeling,reagent and other supplies,salaries and benefits for three technologists,plus all the institutional assessments (e.g., full-cost basis) required to operate a hospital laboratory.,仪器配制及维修、实验室改造等；,试剂及其他消耗品,三位实验员的薪水,所有的评估分析（如,full-cost basis,）需要动用全院系统,分析方法：,t,检验,成本-效益分析（,Analysis of Cost Data）,31,.,基因分型相关费用the cost of equipment,结果：,1、VRE,initial impetus: serious nosocomial problem-VREs emergence,molecular typing results:,a pattern of numerous “mini” patient-to-patient outbreaks of distinct clones,rather than the spread of a single persisting strain,1、VRE,最初调查：,VRE,医院感染严重,分型结果提示：,多型别、小规模（,mini）,病人间流行,而不是一个型别的流行,32,.,结果：1、VRE1、VRE32.,genomic typing:,patient-to-patient transmission;,nosocomial outbreak;,little evidence of horizontal spread,Using this information, we determined what intervention was likely to control an apparent outbreak (,20,).,结果：,基因分型：可将可能的医院感染分组：,病人之间交叉感染(,high conality, 90%),感染爆发(,moderate clonality, 35%-75%),无水平传播,(90%,likely,patient-to-patient transmission,moderate clonality, 35%-75%,possibly,nosocomial outbreak,clonality,90%likelypati,During the last 2 years of this study,25 possible microbial outbreaks were investigated by the typing laboratory,VRE,fluoroquinolone-resistant,P. aeruginosa,MRSA,E. cloacae,C. difficile,.,通过基因分型，共鉴别25起微生物感染爆发,VRE,氟喹诺酮耐药的铜绿,MRSA,阴沟,难辩梭菌,35,.,During the last 2 years of thi,Classic Spread of Nosocomial Infection,VRE: 19 strains, from 16 patients, in a 2-month period;,14 strains: from one of two clones (88%),Indicating:,a high probability of nosocomial spread,Review:,microbiology laboratory: culture requisitions-no close contact.,Patients: existing direct connection between 11/14 patients (,14,).,infection control practices:,aborted the outbreak,典型的医院感染传播,VRE: 19,株, 来自16个病人，2个月时间内,;,其中十四株：为两个型别中的一个型别,(88%),高度提示感染传播,检查分析:,微生物实验室: 培养过程无密切联系,患者：14人中有11人有直接联系,感染控制：中止暴发,36,.,Classic Spread of Nosocomial I,Moderate Likelihood,of Spread of Nosocomial Infections,During a 1-month period, in a special-care unit,invasive infections, caused by five isolates,Klebsiella pneumoniae,S. epidermidis, and,S. hemolyticus,DNA typing indicated 40% to 60% for each of the bacterial species.,patients with genetically identical organisms,occupied adjacent beds.,Erecting a barrier on the unit, along with educating medical staff, halted the spread of these infections (,15,).,较有可能为,NI,传播,1个月时间内，特殊病房,侵入性操作感染：,肺炎克雷伯菌,表皮葡萄球菌,溶血葡萄球菌,40%-60%,clonality,分析：,分离出相同型别菌株的患者病床临近,措施：,病房设立屏障,医护人员教育,结果：感染中止,37,.,Moderate Likelihood of Spread,Outbreaks,not Caused,by Patient-to-Patient Spread,Suspected outbreaks consisting of,four isolates of,K. pneumonia,and,64 strains of,Serratia marcescens,were investigated in the ICUs of two hospitals. Both investigations showed 21% clonality, indicating unlikely patient-to-patient spread.,Investigation suggested suboptimal handling of ventilator equipment, and both outbreaks were stopped by retraining of personnel using this equipment,2,个医院的,ICU,病房,4,株肺克，,64,株粘质沙雷菌,21% clonality,提示：非病人之间传播,indicating unlikely patient-to-patient spread.,调查分析：,机械通气相关操作不规范,措施：规范操作,结果：感染中止,38,.,Outbreaks not Caused by Patien,Pseudooutbreaks,Possible outbreaks occurred in the special-care nursery units of two hospitals, each of which had its own molecular typing section.,seven,S. aureus,strains, and the other of four isolates of gram-negative bacilli.,immediately typed and no (20%) clonality existed.,No interventions were instituted, and the apparent outbreaks were determined to be normal variation in infections (,15,21,).,avoided culture-based surveillance investigation of staff by the state department of health, and the other avoided closing the unit for a 30-day full disinfection and cleaning (done in previous suspected outbreaks).,2个医院，特殊护理病房，每个医院都有自己的分子分型部门,7株金葡,迅速分子分型,rapid typing,no (20%) clonality,没有采取措施,节省：,医护人员携菌情况调查（培养）,关闭病房30天，消毒、清洁,4株,G-,菌,39,.,Pseudooutbreaks2个医院，特殊护理病房，每个医,分型确认后及干预效果,P,=0.000006,LR Peterson,et al, EID, 2001, 7: 306,40,.,分型确认后及干预效果P=0.000006LR Peterso,分型确认后及干预效果,P,=0.002,LR Peterson,et al, EID, 2001, 7: 306,5.79,41,.,分型确认后及干预效果P=0.002LR Peterson5.,nosocomial infection: 3.3%-2.6% (national rate: 4.4%-5%),1,400 fewer patients acquired infections during this time,averting more than 50 expected deaths,Even with endemic VRE, most of our outbreaks involve three or fewer patients (,19,).,医院感染：,3.3%,下降至2.6%(全国医院感染率：,4.4%-5%,),减少1,400的病人感染,死亡：减少了50,VRE：,涉及的病人也比其他医院少,42,.,nosocomial infection: 3.3%-2.6,成本,The mean number of patients with nosocomial infections decreased by 283 per year, a reduction of more than 1,100 inpatient days.,The costs avoided by using this calculation averaged more than $2,150,000/year, based on 1999 dollars.,医院感染患者数量平均每年下降 283，住院天数下降超过1100天,因此节省的费用平均每年超过,$2,150,000（,与1999年相比）,43,.,成本The mean number of patients,Representatives now meet together for 45 minutes each week,For Microbiology, opening the typing laboratory totaled $180,050. By the fifth year, costs in the laboratory section were stable.,The cost for the laboratory, includng three medical technologists, is $400,000 yearly.,Virtually all these costs are borne by the hospital.,小组聚会逐渐转为每周开会，45,min，,讨论,微生物室成立分子分型实验室（设备及人员）的费用为,$180,050.,每年分子分型相关支出为,$400,000,医院承担,44,.,Representatives now meet toget,While such a grant program would cost up to $2 billion each year if all U.S. hospitals participated,the projected reduction in cost of treating nosocomial infections could reach over five times that amount.,a savings of $5.00 for each dollar spent.,假设：美国所有医院,进行基因分型相关费用达到20亿美元,节省下来的治疗医院感染的费用将超过5倍（100亿）！,每使用1美元节省5美元,45,.,While such a grant program wou,Typing time: within 1 week,48 hours.,Lack of clonality: suggests other reasons for the apparent outbreak,antimicrobial-agent use pressure,failure of appropriate nursing-care practices,or simply random variation in the number of infections.,Early knowledge of whether microbial clonality is present or absent focuses the scope of an investigation and facilitates appropriate intervention.,时间：1周，48,h,如没有流行相关线索，可能是其他原因：,抗生素压力,护理操作不当（非感染相关）,仅仅是感染数量的随机变化,早发现、及早确定调查范围、采取合适的干预措施,46,.,Typing time: within 1 week 时间：,cost of rapid detection using the polymerase chain reaction,(PCR),=one day of glove isolation,could be completed in a single 8-hour workday.,As,gene chip,technology moves into clinical use, detecting a large number of resistance determinants soon after a patient is admitted to the hospital should be possible.,PCR,分型,费用=一天的手套费用,8,小时的工作时间内可完成,基因芯片：,大规模,耐药监测,病人入院后即实施,47,.,cost of rapid detection using,分子分型在医院感染中的应用：,technically possible,medically useful,economically justified,48,.,分子分型在医院感染中的应用：technically poss,医院感染的分子流行病学研究方法,分子流行病学方法在医院感染中的应用,医院感染控制的人员安排,成本-效益研究,临床医院感染控制,科研,49,.,医院感染的分子流行病学研究方法临床医院感染控制49.,实验研究：,瑞金医院 04年05年,全耐药鲍曼不动杆菌（,PRAB,） 各个科室的突然增多,经脉冲场凝胶电泳（,PFGE）,证实,烧伤科为单独一个型别,PRAB,科室内流行,除烧伤科以外的其他科室为科室间同一型别,PRAB,流行。,由此可见，分子流行病学方法：,为医院感染控制,提供准确的实验数据,有效判断感染来源和流行趋势,为更好的做好医院感染控制工作打下了基础。,50,.,实验研究：瑞金医院 04年05年50.,Thank You,51,.,Thank You51.,个人观点供参考，欢迎讨论,个人观点供参考，欢迎讨论,