【作者】 敖俊红；

【导师】 郝飞；

【作者基本信息】 第三军医大学 ， 皮肤病与性病学， 2007， 博士

【摘要】 一、研究背景和目的曲霉是广泛存在于自然环境中的一种条件致病菌,与变应性支气管肺曲霉病、曲霉球以及免疫功能低下患者侵袭性曲霉病的发生有关。近年来,随着骨髓及器官移植的广泛开展,广谱抗生素、皮质类固醇激素、免疫抑制剂以及肿瘤药物的大量应用,侵袭性曲霉病发病率明显增加。尽管侵袭性曲霉感染的发生与宿主的免疫状态有关,但医院环境中曲霉含量、曲霉种类以及其变化也间接影响着医源性曲霉感染的发生。有研究资料表明,高危患者病房或医院附近环境的修缮和建筑施工与医源性侵袭性曲霉病的发生具有一定关系,医院环境存在曲霉孢子的污染,同时也证实了大多数免疫抑制患者侵袭性曲霉病的发生与医院环境污染有关。曲霉感染临床表现缺乏特征性,传统的真菌培养和组织病理方法不能满足侵袭性曲霉病的早期诊断。同时,尽管给予较强抗真菌治疗,但侵袭性曲霉病的预后很差,病死率高达50%以上。目前,两性霉素B仍是首选的一线治疗药物,有资料表明伊曲康唑和特比萘芬也可用于侵袭性曲霉病的治疗,但随着抗真菌药的广泛应用,感染菌株对药物的敏感性正逐渐下降,正确合理地选择抗真菌药物及对侵袭性曲霉病的治疗已成为一个突出而且亟待解决的问题。因此,本实验以肝移植病房、脑外和中心ICU病房环境和病房的高危人群为研究对象,对患者体内曲霉存在状况和医院病房环境曲霉分布特点及其影响因素进行监测,采用分子生物学技术追踪患者体内曲霉感染的来源,探讨特比奈芬、伊曲康唑、两性霉素B、氟康唑和氟胞嘧啶单独和联合用药时对环境和患者监测中分离鉴定的曲霉的敏感性,并分析曲霉药敏表型和基因型的关系,为针对性开展预防和治疗曲霉感染提供重要的依据。二、方法和结果1.2005年11月4日-2006年10月23日采用LWC-Ⅰ型离心式空气采样器对肝移植病房、脑外ICU病房和中心ICU病房空气、物表、水源以及外界空气等环境标本进行收集培养和鉴定。结果发现肝移植病房、脑外ICU病房、中心ICU病房及外界空气真菌浓度分别为123.63cfu/m~3、139.90cfu/m~3、7cfu/m~3和214cfu/m~3。中心ICU病房空气真菌浓度明显低于肝移植病房、脑外ICU病房,两者相比差异具有统计学意义(P＜0.01),而肝移植病房、脑外ICU病房空气真菌浓度差异无统计学意义(P＞0.05)。医院病房空气和物表中五种常见真菌依次为青霉、枝孢霉、链格孢霉、曲霉和酵母菌。肝移植病房空气真菌浓度与病房温度和湿度具有相关性,Pearson相关系数分别为0.520、0.637,P＜0.01;脑外ICU病房空气真菌浓度与病房温度和湿度具有相关性,Pearson相关系数分别为0.534、0.573,P＜0.01。肝移植病房、脑外ICU病房和中心ICU病房物表真菌的浓度分别为0.181cfu/cm~2、0.110 cfn/cm~2和0.211cfu/cm~2,不同病区物表真菌污染程度差异无统计学意义(P＞0.05)。病房治疗台面、空调入风口、空调出风口、水龙头表面真菌浓度分别为0.035 cfu/cm~2、0.156cfu/cm~2、0.706cfu/cm~2和0.145cfu/cm~2,病房空调出风口真菌浓度明显高于其他物表真菌浓度,治疗台面真菌浓度明显低于其他物表真菌浓度,差异具有统计学意义(P＜0.01,P＜0.05)。脑外ICU病房和中心ICU病房水源真菌浓度分别为2.9cfu/500ml、2.86cfu/500ml、2.94cfu/500ml,三个病区水源真菌浓度差异无统计学意义(P＞0.05),水源分离的五种常见真菌依次为酵母菌、念珠菌、曲霉、青霉和红酵母。2.对肝移植病房、脑外ICU病房和中心ICU病房环境及高危患者鼻腔、咽部和痰液标本进行监测培养,结果肝移植病房、脑外ICU病房、中心ICU病房空气曲霉浓度分别12cfu/m~3、10.75cfu/m~3和Ocfu/m~3,中心ICU病房空气曲霉浓度明显低于肝移植病房和脑外ICU病房,两者相比差异具有统计学意义(P＜0.05),而肝移植病房、脑外ICU病房空气曲霉浓度差异无统计学意义(P＞0.05)。医院环境中五种常见的曲霉依次为黄曲霉、烟曲霉、黑曲霉、杂色曲霉和棒曲霉,肝移植病房和脑外ICU病房空气曲霉浓度与与活动人员具有相关性,Pearson相关系数分别为0.417、0.467,P＜0.05。肝移植病房、脑外ICU病房和中心ICU病房物表曲霉浓度分别为0.02cfu/cm~2、0.010cfu/cm~2和0.037cfu/cm~2,中心ICU病房物表曲霉污染程度明显高于肝移植病房和脑外ICU病房,差异具有统计学意义(P＜0.05),而肝移植病房与脑外ICU病房物表曲霉污染程度差异无统计学意义(P＞0.05)。病房治疗台面、空调入风口及出风口、水龙头曲霉浓度分别为0.006cfu/cm~2、0.013cfu/cm~2、0.122cfu/cm~2和0.013cfu/cm~2,ICU病房空调出风口曲霉污染程度较严重,治疗台面曲霉染程度较轻。肝移植病房、脑外ICU病房和中心ICU病房水源曲霉浓度分别为0.68cfu/500mL、0.5cfu/500ml和0.34cfu/500ml,三个病区水源曲霉浓度差异无统计学意义。水源分离的常见曲霉依次为黄曲霉、黑曲霉、烟曲霉和杂色曲霉。从5例高危患者的鼻腔、咽部和痰液共分离出33株黄曲霉和3株烟曲霉,采用随机扩增多态性DNA(random amplification of polymorphic DNA,RAPD)分析方法对分离自环境和患者体内的黄曲霉进行基因分型研究,结果发现脑外ICU 2例患者体内分离的黄曲霉与病房环境分离的黄曲霉基因型相同,中心ICU病房3例患者体内分离的黄曲霉与病房环境分离的黄曲霉基因型均不相同,但2例患者体内分离的黄曲霉基因型相同。3.应用美国临床实验室标准化委员会制定的M38-A方案探讨了两性霉素B(AmB)、特比奈芬(TBF)、伊曲康唑(ICZ)、氟胞嘧啶(5-FC)和氟康唑(FCZ)在单独和联合用药时对曲霉的敏感性,单独用药时AmB、TBF和ICZ取100%生长抑制为最小抑菌浓度(MIC),5-FC和FCZ取50%生长抑制为最小抑菌浓度,联合用药时取100%生长抑制为最小抑菌浓度,以部分抑菌浓度(FIC)来判定药物间的相互作用,药物间的相互作用解释为FIC≤0.5为协同作用,0.5＜FIC≤1为相加作用,1＜FIC≤2为无关作用,FIC＞2为拮抗作用。结果发现特比奈芬对烟曲霉的MIC值(MIC=1.578μg/ml)明显高于黄曲霉和黑曲霉,伊曲康唑对黄曲霉的MIC值较低(MIC=0.104μg/ml),两性霉素B对黑曲霉的MIC值较低(MIC=0.094μg/ml),而对黄曲霉MIC值较高(MIC=1.809μg/ml),差异具有统计学意义。氟康唑和氟胞嘧啶对曲霉MIC值较高(MIC分别为25.77μg/ml和3.1μg/ml)。伊曲康唑、两性霉素B、氟康唑、氟胞嘧啶均存在耐药菌株,且大部分为临床分离株。特比奈芬联合伊曲康唑、两性霉素B联合伊曲康唑对曲霉菌属产生较好的协同相加作用(FIC=0.13～2.5,FIC=0.25～3),两性霉素B联合特比奈芬对曲霉主要表现为相加作用(FIC=0.28～4.99),氟胞嘧啶和氟康唑与其他药物联合应用对曲霉菌属表现为拮抗和无关作用(FIC=0.28～16.7,FIC=0.25～16),仅对少数菌株表现为协同和相加作用。应用RAPD技术对同种曲霉进行基因分型,分析曲霉的药敏表型和基因型的关系,结果发现不同基因型的烟曲霉和黑曲霉分别对特比奈芬和两性霉素B敏感性存在差异,而不同基因型黄曲霉对5种抗真菌药物敏感性不存在差异。三、结论1.真菌存在于医院病房空气、物表以及水源中,空气中真菌全年均有分布,且5～6月、9～10月为2个高峰期,室内外峰期基本一致。病房温度和相对湿度与病房空气真菌浓度具有相关性。脑外ICU病房空气真菌污染程度较高,中心ICU病房空气真菌污染程度较低;病房空调出风口真菌污染程度较为严重,治疗操作台面真菌污染程度较低。2.肝移植病房、脑外ICU病房和中心ICU病房存在不同程度曲霉污染,脑外ICU2例患者体内分离的黄曲霉和环境中分离的黄曲霉基因型相同,推测患者感染的黄曲霉可能来源于医院环境中。3.单独用药时不同抗真菌药物对曲霉菌种的敏感性具有差异。特比奈芬联合伊曲康唑、两性霉素B联合伊曲康唑在体外对曲霉具有协同和相加作用,其效果优于其他药物的联合应用。曲霉的基因型与其对抗真菌药物敏感性具有相关性。更多还原

【Abstract】 Background and ObjectivesAspergillus is ubiquitous in the environment and capable of causing allergic, aspergilloma and invasive aspergillosis(IA) in immunocompromised patients.The incidence of life-threatening invasive aspergillus infection has been increasing with bone or organ transplant,the using of high-dose corticosteroids or immunosuppressive therapies etc. Although the immune status of host is thought to be the major contributor to the establishment of infection,air Aspergillus load and its fluctuations in the hospital environment are expected indirectly to influence the incidence of hospital-acquired Aspergillus infection,as well as the predominant specie.Much of our current understanding about the transmission of IA is based on information gathered from outbreak investigations. Nosocomial outbreaks of IA have become a well-recognized complication of construction or renovation work in or near hospital wards in which high-risk patients are housed.These reports have highlighted the fact that hospital air is often contaminated with Aspergillus spores,and they have contributed to the current perception that most cases of IA in immunocompromised persons are hospital acquired.Invasive Aspergillus infections are hard to diagnose at an early state because their clinical presentation is usually non-specific. The traditional diagnostic evidence derived from mycological cultivation or histological demonstration of fungi within tissue samples is difficult to obtain.Despite appropriate antifungal therapy,the prognosis of IA remains very poor and the overall mortality is higher than 50%.Amphotericin B is the first agent for the initial treatment of Aspergillus infection.Some data indicate that itraconazole and terbinafine are two agents licensed for treatment of Aspergillus infection.But with the resistance of Aspergillus increasing,the treatment of choice for infected patients has become outstanding problem.In order to prevent and cure the invasive Aspergillus infection,we surveillance the Aspergillus load and species distribution in environment and patients of transplantation department and intensive care unit in Southwest hospital,and the relationship with season, temperature,humidity,ventilation and personnel activities are also analyzed.Aspergillus isolated from environment and patients were genotyped to determine the origin of infection by random amplification of polymorphic DNA(RAPD).We also investigate the activities of amphotericin B(AraB),itraconazole(ICZ),terbinafine(TBF),fluconazole(FCZ) and 5-flucytosine(5-FC) alone and interaction in combination against Aspergillus and analyze the relationship of genotype and antifungal susceptibility.Methods and Results1.Air,surfaces and tap water were sampled twice a month from liver transplantation department(LTD),cerebral surgery intensive care unit(CSICU) and central intensive care unit(CICU) using LWC-1 air-samplers.The results showed that air fungal load was 123.63cfu/m~3,139.90cfu/m~3,7cfu/m~3 and 214cfu/m~3 in LTD,CSICU,CICU and outdoor respectively.Air fungal load was higher in CSICU and LTD than CICU(P＜0.01).The five most prevalent fungi collected from air and surface was Penicillium spp.,Cladospcrium spp.,Alternaria spp.,Aspergillus spp.and Saccharomyces spp.in turn.The fungal load in CSICU and LTD was correlated with the average temperature and the average humidity,but the correlation between air fungal load and personnel activities weren’t observed.The fungal load of surfaces was 0.181cfu/cm~2,0.110cfu/cm~2 and 0.211cfu/cm~2 in LTD, CSICU and CICU respectively.The difference of fungal contamination between different departments was not statistically significant(P＞0.05).The fungal load was 0.035cfu/cm~2, 0.145cfu/cm~2,0.156cfu/cm~2 and 0.706cfu/cm~2 at treatment unit,tap,air conditioning vent and inlet respectively.The fungal load of air conditioning vent was higher than other surfaces and tap was lower than other surfaces.The fungal load of water was 2.gcfu/500ml, 2.86cfu/500ml and 2.94cfu/500ml in LTD,CSICU and CICU respectively.The five most prevalent fungi collected from water were Saccharomyces spp.,Candida spp.,Aspergillus spp.,Penicillium spp.and Rhodotorula spp.in turn.2.Air,surfaces and tap water were sampled twice a month from LTD;CSICU and CICU.Clinic sample of nose,pharynx and sputum collected from patients.The results showed that mean total Aspergillus was 12cfu/m~3,10.75cfu/m~3,0cfu/m~3 and 20cfu/m~3 at LTD,CSICU,CICU and outdoor respectively.Air fungal load was lower in CICU than others(P＜0.05).The five most prevalent Aspergillus species collected inside the hospital were Aspergillus flavus,Aspergillus fumigatus,Aspergillus niger,Aspergillus versicolor and Aspergillus clavatus.The fungal loads in CSICU and LTD were correlated with personnel activities,but the the correlation between air fungal load and humidity or temperature weren’t observed.The Aspergillus loads of surfaces were 0.02cfu/cm~2,0.010cfu/cm~2 and 0.037cfu/cm~2 in LTD,CSICU and CICU respectively.The Aspergillus load of surfaces was higher in CICU than in others(P＜0.05).The Aspergillus loads were 0.006cfu/cm~2,0.013cfu/cm~2, 0.013cfu/cm~2 and 0.122cfu/cm~2 at treatment unit,trap,air conditioning vent and inlet respectively.The Aspergillus loads of air conditioning vent was higher than other surfaces and tap were lower than other surfaces.The Aspergillus loads of water were 0.68cfu/500ml, 0.5cfu/500ml and 0.34cfu/500ml in LTD,CSICU and CICU respectively.The most prevalent fungi collected from water were Aspergillus flavus,Aspergillus niger,Aspergillus fumigatus and Aspergillus versicolor in turn.Thirty-three Aspergillus flavus and three Aspergillus fumigatus species were isolated from nose,pharynx and sputum of five patients.Aspergillus flavus isolated from environment and patients were genotyped to determine the origin of infection by random amplification of polymorphic DNA(RAPD).RAPD analysis demonstrated that strains isolated from patients in CSICU were identical to environmental strain.Strains isolated from patients in ICU were different to environment strain,but strains isolated from two patients were identical.3.NCCLS M38-A protocol was employed to investigate the activities of amphotericin B(AraB),itraconazole(ICZ),terbinafine(TBF),fluconazole(FCZ) and 5-flucytosine (5-FC) alone and interaction in combination against Aspergillus.In test alone,the MIC endpoints were determine of AraB,TBF,ICZ as 100%growth reduction compared with turbidity produced by the control wall,FCZ and 5-FC were as≥50%growth reduction,but 100%growth reduction when they were given in combination.Drug interactions were classified on the basis of the fractional inhibitory concentration(FIC) index.The interaction was defined as synergic if the FIC index was 0.50,additive if the FIC index was＞0.50～1.0, indifferent if the FIC index was＞1.0～2.0 and antagonistic if the FIC index was＞2.0.The results showed that MIC of TBF was highest for Aspergillus fumigatus(1.578μg/ml).MIC of ICZ was lowest for Aspergillus flavus(0.104μg/ml).MIC of AraB was lowest for Aspergillus niger(0.094μg/ml) and highest for Aspergillusflavus(1.809μg/ml).MIC of FCZ and 5-FC were much higher than other drug for Aspergillus(25.77μg/ml,3.1μg/ml respectively).Much of clinic isolates were resistant to FCZ,ICZ,AmB and 5-FC.The combination of ICZ-TBF and ICZ-AmB were synergic or additive against most Aspergillus (FIC=0.13～2.5,FIC=0.25～3 respectively).The combination of TBF and AmB were additive against most Aspergillus(FIC=0.28～4.99).The combination of FCZ and 5-FC with other drugs were antagonistic or indifferent(FIC=0.28～16.7,FIC=0.25～16 respectively).The relationship between genotype of Aspergillus and its sensitivity to antifungal drugs was analysed by dendrogram according to RAPD patterns.The results showed that there were differences among sensitivity to TBF of the genotypes of Aspergillus fumigatus and to AmB of the genotypes of Aspergillus niger.But no statistically difference was found among antifungal angents of the genotypes of Aspergillusflavus.Conclusions1.It demonstrated the fungus was found in the environment of the hospital including air,surface and water.The air fungal load varies throughout the year.The crest-time was May to June and September to October.The correlation between air fungal load and temperature,humidity was observed.Air fungal load was lower in CICU and higher in CSICU.2.Aspergillus contamination was found in LTD,CSICU and CICU.Clinic and environmental strain from CSICU have identical genotype;the infection may be from hospital environment.3.The sensitivity of antifungal agents were different when test alone to different species of Aspergillus.The combination of ICZ-TBF and ICZ-AmB in vitro displayed a potent synergic or additive against most Aspergillus compared with the combination of other drugs.The correlation between sensitivity to antifungal drugs and genotypes of Aspergillus was observed.更多还原