1、1,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,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,3,4,消化、释放出DNA,PFGE原理,5,6,7,8,9,时间消耗 从
2、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,10,11,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,12,PFGE同源性分析,13,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
3、,14,2、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910bp 扩增条件：“非严格性” 退火温度一般较低,2535,15,16,17,18,19,Special IssueNew Technology for Detecting Multidrug-Resistant Pathogens in the Clinical Microbiology Laboratory Lance R. Peterson* and Gary A. Noskin* *Northwestern Me
4、morial Hospital and Northwestern University Medical School, Chicago, Illinois, USA,EID, 2001, 7: 306,20,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,21,分型确认后及干预效果,P=0.002,LR Peterson et al, EID, 2001, 7: 306,5.79,22,指标,t
5、he 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）,23,24,25,Two interventions： a molecular typing laboratory a weekly plan
6、ning 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,26,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 trend
7、s 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 clinic
8、al microbiology,27,28,实验方法,REA analysis restriction of genomic DNA with conventional electrophoresis DNA限制性酶切,29,30,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
9、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,31,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
10、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(20% clonality). 在此基础上，决定采取哪种控制措施,32,similarity,33,During the last 2 years of this study,25 possible microbial outbreaks were investigated by the typing laboratory VRE, fluoroquinolone-resistant
11、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,34,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: microb
12、iology 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,35,Mod
13、erate 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 iden
14、tical 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,36,Outbreaks not Caused by Patie
15、nt-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 handl
16、ing 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,37,38,分型确认后及干预效果,P=0.000006,LR Peterson et al, EID, 2001, 7: 3
17、06,39,分型确认后及干预效果,P=0.002,LR Peterson et al, EID, 2001, 7: 306,5.79,40,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
18、 (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,41,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
19、 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）,42,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 labora
20、tory, 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，45min，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,43,While such a grant program would cost up to $2 billion each year if all U.S. hospitals participated
21、, 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,44,Typing time: within 1 week 48 hours. Lack of clonality: suggests other reasons
22、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 in
23、tervention.,时间：1周，48h 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,45,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
24、 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,46,分子分型在医院感染中的应用：,technically possible medically useful economically justified,47,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,48,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。,Thank You,
