【作者】 胡洪波；

【导师】 黄汉菊；

【作者基本信息】 华中科技大学 ， 病原生物学， 2010， 博士

【摘要】 呼吸机相关性肺炎(Ventilator-associated pneumonia, VAP)是指“患者在建立人工气道(气管插管或切开)及机械通气48小时以后或撤机拔管后48小时以内所发生的医院获得性肺炎。”患儿一旦发生VAP,易造成脱机困难,住院时间延长,医疗费用增加,严重者甚至可能威胁生命,造成死亡,是近年来全球新生儿重症监护室(NICU)患儿死亡的主要原因。VAP的危害已引起了广大临床医务人员和学者的广泛关注,但VAP的发病机理尚不十分清楚,归纳起来主要包括以下两个方面：(1)患儿防御机制受损；(2)病原侵袭机会增加。病原通过内源性途径和外源性途径侵袭下呼吸道,内源性途径可能是由于口咽部定植菌和胃肠反流内容物的吸入造成,外源性途径可能是外界环境污染或细菌生物被膜脱落碎片的吸入。铜绿假单胞菌(Pseudomonas aeruginosa, PA)是引起迟发性呼吸机相关肺炎(通气时间>4天)的常见病原体之一,由该菌引起的感染在NICU有很高的发病率和死亡率。目前,大部分机械通气患儿肺部感染被认为是内源性感染,但在治疗、诊断和预防过程中,因为医务人员水平传播造成的感染也不容忽视。关于机械通气患儿PA定植或／和感染来源、途径及相关危险因素的研究,国内外少有报道。只有在充分了解VAP致病机理的基础上,才能够采取合理、有效的措施,预防和减少VAP的发生。目的1.了解NICU中机械通气患儿PA定植或／和感染的临床特点和感染途径。2.检测PA临床株的耐药基因,了解PA临床株与环境株的耐药特点。3.了解机械通气患儿PA临床株及NICU环境株的分型,调查VAP患儿PA感染来源(外源性感染,内源性感染)4.探讨NICU中机械通气患儿PA定植或／和感染的危险因素。方法选取2009年1月至2009年6月在我院新生儿科重症监护室治疗,机械通气治疗时间大于4天的临床病例为研究对象。所有机械通气患儿在上机前取咽拭、气管分泌物、胃液及直肠拭子送检,以后每周采样2次直至撤机。同时,每5天采集NICU环境标本一次,当日送检。采用ATB细菌鉴定分析系统,对临床分离细菌进行鉴定,并采用K-B法选取头孢他啶(CAZ)、头孢吡肟(FEP)、哌拉西林(PIP)、哌拉西林／他唑巴坦(TZP)、阿米卡星(AMK)、头孢哌酮(CFP)、头孢哌酮／舒巴坦(SPF)、苯唑西林(MEZ)、环丙沙星(CIP)、亚胺培南(IMP)、美罗培南(MER)、左氧氟沙星(LVX)12种常用抗菌药物进行体外药物敏感试验,判定标准参照美国国家临床实验室标准委员会(NCCLS)2006年公布的细菌药敏试验标准。对耐药种类≥3种的临床标本进行β-内酰胺酶耐药基因的检测。通过细菌耐药谱、脉冲场凝胶电泳(PFGE)及低频限制性位点聚合酶链反应(IRS-PCR)对PA进行分型,条带判读及分型依据参照Tenover等制定的分型标准。选取PA定植或／和感染患儿为病例组,同期收治的同病区,无PA定植及细菌感染的机械通气患儿为对照组。单因素分析,计数资料采用x2检验(部分为校正x2值),计量资料用独立样本资料t检验。计算比值比(Odds Ratio, OR)和95%的置信区间(CI)。经过单因素分析,P值<0.1的变量纳入进一步的Logistic回归分析。采用基于最大似然估计的前进法进行非条件Logistic回归分析,筛选机械通气患儿PA定植或／和感染的独立危险因素,P值<0.05的变量认为有统计学意义。所有的统计均使用SPSS12.0统计学软件完成。结果1.2009年1月至2009年6月,共129例患儿通气时间>4天,PA定植41例(31.8%),10例(7.8%)发生在气管插管前,31例纳入研究对象。8例发展为VAP, PA VAP发生率为6.2%。2.31株PA对哌拉西林／他唑巴坦、阿米卡星、头孢哌酮／舒巴坦、环丙沙星、亚胺培南、美罗培南和左氧氟沙星的敏感率较高,均>70%；17株PA环境株除苯唑西林外,对其他抗生素均较敏感。对20株耐药种类≥3种的临床标本耐药基因检测显示,100%的菌株β-内酰胺酶编码基因检测阳性,其中TEM、CRAB、VIM、IMP和FOX的检出率分别为55.0%、25.0%、15.0%、15.0%和5.0%,未检出其他耐药基因。3.23例PA定植患儿中,定植部位以上呼吸道定植为主,占18例(78.3%)。8例VAP患儿,早期均发生上呼吸道定植,其中3例伴有消化道定植,PA定植次序均为上呼吸道→下呼吸道,无消化道→上呼吸道→下呼吸道的逆向定植次序。4.31例患儿共分离出54株PA临床株,同期分离17株PA环境株。根据PFGE分型,分为17种基因型；根据IRS-PCR分型,分为17种基因型。两种分型结果完全一致。8例VAP患儿中,4例(50%)为内源性感染,4例(50%)PA分离株分型与其他患儿分离株或环境株一致,为外源性感染。5.出生体重<1500克(OR,6.830)；机械通气时间≥8天(OR,3.324)；使用氨苄类青霉素(OR,3.631)以及使用二代头孢(0R,4.550)为机械通气患儿PA定植或／和感染的独立危险因素(P<0.05)。结论针对机械通气患儿PA定植、感染的流行病学特点及相关危险因素,应该采取综合防治措施预防VAP的发生：(1)做好围生期保健,降低极低出生体重儿、早产儿的出生率；(2)积极治疗患儿原发性疾病,尽量缩短机械通气时间,及时撤机；(3)加强NICU的管理,增强医务人员的消毒意识,做好NICU传染病区的消毒隔离,在接触患儿前后做好手部的清洁与消毒工作；(4)加强呼吸机管道、雾化器、氧气湿化瓶的消毒灭菌,每7天更换呼吸机管道一次。(5)加强对NICU的环境卫生(包括空气标本、物体表面、呼吸机管道、消毒液、医务人员的手)监测；(6)加强对机械通气患儿呼吸道及消化道的定植菌监测；7)尽量避免盲目性或经验性的应用多种广谱抗生素,根据药敏试验结果合理选用抗生素。更多还原

【Abstract】 Ventilator-associated pneumonia (VAP) is one of the most serious complications in mechanically ventilated patients that develop≥48 h after the patient has been placed on mechanical ventilation (MV). Pseudomonas aeruginosa (PA) is the most common isolates in late onset pneumonia which developed beyond the 4th day of ventilation, associated with the worst morbidity and mortality rates in the neonatal intensive care units (NICU). The pathophysiology of a patient’s pulmonary colonization with PA is still unclear:its main origin seems to be endogenous but the contaminated device, environment, and the colonized patients have been clearly shown to be a source and to be involved in horizontal transmission. PA VAP has rarely been reported in NICU. Based on understanding of colonization pathogenesis, rational strategies for nosocomial pneumonia prophylaxis can be instituted. The routes and patterns of colonization or infection with PA and associated risk factors, essential to design appropriate prevention strategies, has rarely been exploited by active surveillance studies.Objective 1.To investigate the respective contribution of endogenous and exogenous transmission of PA in the respiratory colonization or/and infection in the mechanically ventilated patients at a NICU;2. To investigate the distribution of drug resistance of PA;3. To identify routes of lung infection with PA;4. To assess risk factors for colonization or respiratory infection with PA; These findings may be important for the design preventive strategies from PA colonization and pulmonary infection in NICUMethodsA 6-months surveillance prospective survey was performed from January 2009 through June 2009. Samples from oropharyngeal swab, tracheobronchial aspirates, gastric aspirate, and rectal swab were obtained in each patient just before ventilation and then two times per week. Surveillance cultures for the presence of PA from environmental surfaces of the NICU were taken once every five days during the study period. To analyze the predisposing factors for developing VAP due to PA, the following variables were recorded:demographic characteristics, history of prior hospitalizations and antimicrobial use, prior barbiturate use, diagnosis, clinical features, prior trauma or surgery, daily ventilator settings, the duration of MV prior to the development of VAP or colonization, and respiratory procedures during ICU stay. For each colonized or infected patient, a chronological analysis of the isolation was performed. Antibiotic susceptibility was determined by the disk-diffusion method and interpreted according to NCCLS guidelines. The following antibiotics were tested: ceftazidime (CAZ), cefepime (FEP), piperacillin (PIP), piperacillin/tazobactam (TZP), amikacin (AMK), cefoperazone (CFP), cefoperazone/sulbactam (SPF), mezlocillin (MEZ), ciprofloxacin (CIP), imipenem (IMP), meropenem (MER) and levofloxaxin(LVX).Antibiotyping, IRS-PCR and PFGE indicated the epidemiological relationship. Patterns were analyzed as recommended by Tenover et al. Infections showing clinical symptoms and positive cultures at 48 hours or more after birth were defined as NI. Colonization was defined as the isolation of PA from specimens taken from any body site studied without clinical or bacteriologic evidence of infection. To determine the risk factors associated with PA colonization/infection in the mechanically ventilated patients, a case-control study was carried out, with cases involving PA colonized and/or infected neonates and controls, those without colonization by PA, and infection by any microorganism. Potential risk factors were analyzed by univariate and multivariate analysis. To test the independence of the risk factors for PA colonization/infection, the significant variables(p<0.1) in the univariate analyses were entered into a multivariate logistic regression model with forward selection of independent variables. The software package SPSS12.0 was used for the analysis.Results1. During the study period of the 129 patients on mechanically ventilation was more than four days,41 (31.8%) PA colonization proved, among which 10 (7.8%) were already colonized before ventilation. Thus 31 (24.0%) patients were classified as acquired colonized and included in this study.8 patients presented VAP caused by PA. The incidence of PA VAP on the unit was 6.2%. The mean period of MV prior to the VAP onset was 9±3.4 days.2. The sensitivity to amikacin, levofloxacin, ciprofloxacin, piperacillin/tazobactam, cefoperazone/sulbactam, imipenem and meropenem was respectively over70.0%. PAE was inferior sensitivity to piperacillin, mezlocillin, cefoperazone and ceftazidime. The positive rates of gene encoding extended-spectrumβ-lactamase TEM, CRAB, VIM, IMP and FOX were 55.0%,25.0%,15.0%,15.0% and 5.0%; other P-lactamase genes were absent in all isolates.2. In our study 18 of 23 patients (78.3%) had colonization of the upper respiratory tract. The respiratory tract in 8 patients with VAP had been colonized previously by the same strain; simultaneous digestive tract colonization was found in three cases. Neither the stomach nor the rectum as the only initial site of colonization in any case was found.3. Molecular characterizations of 71 PA isolates including 54 clinical strains and 17 environmental strains were performed by PFGE and IRS-PCR. Genotyping analysis 17 unrelated patterns were revealed by PFGE and 18 by IRS-PCR (Table 2).16 (69.6%) patients with colonization of the respiratory tract come from other patients or environmental surfaces were considered exogenous, whereas among strains causing pulmonary infection,4 (50%) strains were of exogenous.4. Birth weight<1500g (OR,6.830), MV≥8 days (OR,3.324), previous ampicillin group (OR,3.631) and second generation cephalosporins use (OR,4.550) were independently associated with PA colonization/infection (p<0.05).ConclusionsOur results confirm the upper respiratory tract act as an important reservoir of PA colonization and infection in the mechanically ventilated patients and emphasize the importance of exogenous acquisition of PA. A combination of early identification and eradication of airways colonization by PA plus infection control measures may be the basis to prevent pulmonary infection.更多还原