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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. 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;. - PowerPoint PPT Presentation
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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
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。
3
微生物室在医院感染中的作用
accurately identifying nosocomial pathogens
detecting unexpected antimicrobial-drug resistance
epidemiologic typingepidemiologic 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
4
PFGE(PFGE( 脉冲场凝胶电脉冲场凝胶电泳泳 ))
RAPD(RAPD( 随机扩增随机扩增DNADNA 多态性多态性 ))
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
5
1 、脉冲场凝胶电泳 (PFGE)
制备琼脂糖栓块
制备琼脂糖栓块
消
化消
化酶 切酶 切
脉冲电泳
脉冲电泳
结果解释
结果解释
6
消化、释放出DNA
PFGE 原理
7
Enzyme 酶切
PFGE 原理
约 5 ~ 20 个、长度 10 ~ 800kb 的大片段 DNA
8
Tenover FC.et al.J Clin Microbiol,1995;33(9):2233 ~ 2239
9
脉冲电泳
PFGE 原理
10
原理:
DNA
常规凝胶电泳
脉冲场凝胶电泳
11
时间消耗时间消耗 从分离菌株到出结果平均 从分离菌株到出结果平均 2.2.
5 5 天天 标本准备、细胞裂解--第标本准备、细胞裂解--第
一天一天 酶切--第二天酶切--第二天 染色、拍照--第三天染色、拍照--第三天
PFGE 原理
染色、拍照
12
13
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 2 - 3 个带的认为亲缘关系密切,相差 4 - 6 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
14
PFGE 同源性分析
Dice (Opt:1.50%) (Tol 1.5%-1.5%) (H>0.0% S>0.0%) [0.0%-100.0%]
PFGE - XbaI
100
8060PFGE - XbaI PCR Testing
TE
MP
CR
SH
VP
CR
CT
XP
CR
OX
AP
CR
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
EEX.0042
EEX.0044
EEX.0045
EEX.0038
EEX.0038
EEX.0038
EEX.0013
EEX.0040
EEX.0015
01A
07A
07A
01A
01A
01A
01A
01A
01A
15
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
16
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 9 ~10bp
扩增条件:“非严格性” 退火温度一般较低 ,25 ~
35℃
17 ESHWAR MAHENTHIRALINGAM.et al.J Clin Microbiol,1996;34(5):1129 ~ 1135
18 ESHWAR MAHENTHIRALINGAM.et al.J Clin .et al.J Clin Microbiol,1996;34(5):1129Microbiol,1996;34(5):1129 ~~ 11351135
19 ESHWAR MAHENTHIRALINGAM.et al.J Clin .et al.J Clin Microbiol,1996;34(5):1129Microbiol,1996;34(5):1129 ~~ 11351135
20
分子分型在医院感染控制中的作用
21
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
22
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
23
分型确认后及干预效果
P=0.002
LR Petersonet al, EID, 2001, 7: 306
5.79
24
指标
1. the total number of nosocomial infections per 1,000 patient days每千住院日医院感染数
2. 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)
25
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
26
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。
27
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
28
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
29
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
任何时候任何时候工作小组要求进行基因分型,临床微生物实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验室即将菌株提供给分子分型部门分子分型部门并执行并执行
30
实验方法
REA analysisREA analysis
restriction of genomic DNA with
conventional electrophoresis
DNA 限制性酶切
31
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 )
32
结果: 1 、 VRE initial impetus: serious
nosocomial problem---VRE's 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
33
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 (<20%
clonality). 在此基础上,决定采取哪种
控制措施
34
high conality,
>90%
likely patient-to-patient
transmission
moderate clonality, 35%-75%
possibly nosocomial outbreak
clonality,
<20%
unlikely little evidence of horizontal
spread
similarity
35
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
36
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
37
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
38
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
39
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
40
分型确认后及干预效果
0
0.5
1
1.5
2
2.5
3
3.5
4
NI/100 Patients
Typing Intervention
1993 1994 1995 1996 1997 1998 1999
3.3
2.56
P=0.000006
LR Petersonet al, EID, 2001, 7: 306
41
分型确认后及干预效果
P=0.002
LR Petersonet al, EID, 2001, 7: 306
5.79
42
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
43
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)
44
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, 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
45
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
46
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, 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
47
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
48
分子分型在医院感染中的应用:
technically possible
medically useful
economically justified
49
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
50
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。