肝病患者真菌的感染特点和质谱快速鉴定研究
发布时间：2018-08-08 12:34
【摘要】：目的真菌是广泛分布于自然界的真核生物,已被发现的约10万种,其中对人类具有致病性约300种,不同种真菌的生长特性、临床特点及耐药性不同。由于抗菌药物和免疫抑制剂的广泛应用、放化疗治疗的增加、器官移植及介入技术操作的开展,导致机体免疫功能低下人群增加,使很多机会致病真菌引起感染,成为免疫功能低下患者的重要死亡原因。另外,肝病患者由于蛋白低、单核巨噬细胞系统受损、白细胞粘附趋化和吞噬能力降低、肠道内菌群失调等原因成为易感染真菌感染的易感人群,真菌感染的发病率和病死率呈逐年上升趋势,引起临床的高度关注。同时,随着抗真菌药物的大量应用,真菌的耐药性也日益突出,为临床的经验用药提出了挑战。快速明确感染真菌的特点及耐药状况,成为临床救治的关键。真菌的特点是生长缓慢,传统的培养鉴定方法所需时间较长,一般3-7天,且日益多样化的真菌,使鉴定难度不断增加均限制了临床的早期诊断和针对性治疗。基质辅助激光解吸电离飞行时间质谱(Matrix-Assisted Laser-Desorption/Ioni zation Time of Flight Mass Spectrometry,MALDI-TOF MS)是近年快速发展起来的一种新兴的诊断技术,可以通过直接检测生物标志物(蛋白)来鉴定细菌、分枝杆菌、病毒等,具有快速、准确、简便、成本低的特点。但它对真菌的鉴定评价说法不一,主要原因真菌的细胞壁厚,前处理方法不成熟导致鉴定结果的差异。为此本研究通过把质谱仪鉴定真菌的结果与生化鉴定、分子生物学等技术鉴定结果比对,探索质谱技术快速鉴定真菌的方法,评价其真菌鉴定能力,为临床快速诊断真菌感染提供可靠的依据;同时对我院肝病患者真菌感染的临床资料和药敏情况进行统计分析,找到肝病患者真菌感染的病原组成特点及增长趋势,分析其对不同种类抗真菌药物的耐药性,更好的指导临床合理的应用抗真菌药物治疗,达到有效控制真菌感染的目的。方法收集我院2011年-2014年分离自临床肝病住院患者的2236株真菌的临床资料(7天内分离自同一患者相同部位的真菌不重复统计),对其易感人群、感染部位及菌群分布等特点进行统计分析。用氟康唑、两性霉素B、伊曲康唑、伏立康唑对2236株真菌进行K-B法药敏试验,统计分析其耐药性。用布鲁克公司的Microflex型质谱仪对其中327株真菌菌株进行快速鉴定,结果与VITEK-2(酵母样真菌)和显微镜检查(丝状真菌)的鉴定结果比对,有差异的用分子生物学方法确认鉴定。结果从我院近四年肝病患者标本分离真菌的情况分析,真菌感染多发于男性患者,男女比例为1.73:1,平均年龄为54±14.79岁。肝病患者真菌感染更易发生在肝硬化失代偿期、肝癌及重症肝炎患者,其中肝硬化失代偿期的患者所占比例为69.65%,肝癌患者占16.97%。真菌的分离数量从2011年的61株,急剧增长到2014年的1222株,增长了19.03倍。四年共分离真菌2236株,酵母样真菌1889株,占84.48%,丝状真菌347株,占15.52%;酵母样真菌的年分离率占绝对优势,2011年为63.93%,2012年为85.42%,2013年为84.92%,2014年为85.02%。真菌分离的种类也由2011年的15种增加到了目前的近30种。在众多真菌种类中,居首的为白假丝酵母菌(52.64%),其次为热带假丝酵母菌(13.86%)和烟曲霉菌(12.03%)。真菌感染部位多发于呼吸道,占66.41%,其中酵母样真菌占52.24%,丝状真菌占14.18%;呼吸道分离的真菌种类以白假丝酵母菌为主,占35.15%,其次为烟曲霉菌占11.09%。腹水和引流液感染是肝病患者真菌感染的特点,分离率为13.91%,感染种类以白假丝酵母菌为主(8.32%)。从我院近四年的真菌药敏结果分析,丝状真菌的耐药率普遍高于酵母样真菌(p0.05),酵母样真菌对氟康唑、两性霉素、伊曲康唑和伏立康唑的耐药率分别为11.96%,2.54%,1.96%和0.69%;丝状真菌对氟康唑、两性霉素、伊曲康唑和伏立康唑的耐药率分别为95.97%,17.29%,6.05%和4.61%。本研究对327株真菌的质谱鉴定结果显示,依照其评分标准质谱仪鉴定酵母样真菌评分达到种水平(评分2.0)的鉴定率为90.31%,达到属水平(评分1.7)的为98.68%。丝状真菌评分达到种水平的鉴定率为74%,达到属水平的为94%。对临床常见的曲霉菌的鉴定正确率可以达到96.74%。质谱仪还能从种内对真菌进行更细致的分型,例如可准确的将近平滑假丝酵母菌,似平滑假丝酵母菌和拟平滑假丝酵母进行区分。对单一种类真菌感染的阳性培养瓶,质谱从培养瓶中直接提取菌种蛋白鉴定的结果与培养后用菌落鉴定的结果相同,这可以把体液真菌感染的鉴定时间提前24h。本研究通过对现有各种真菌鉴定方法用时的比较,发现质谱仪单株菌鉴定用时仅需20min,并且可同时检测多株菌,是目前真菌鉴定用时最短,操作也较简便的方法,为临床真菌感染的诊断和治疗争取了时间。结论肝病患者真菌感染的数量和种类有逐年上升的趋势,临床要特别重视此类患者腹水、引流液等无菌体液的感染,加强对各种留置管的护理,对各种介入性治疗要严格无菌操作。应根据微生物实验室的菌种鉴定和药敏结果合理进行抗真菌治疗,并且要考虑到药物对肝脏的毒性,慎重选择抗真菌药物。质谱仪在鉴定真菌的种属水平上都达到了理想的结果,尤其鉴定酵母样真菌和曲霉菌的能力更为突出,完全满足临床微生物实验室真菌检测的需求。质谱仪在真菌感染诊断方面有广阔的临床应用前景,对于不常见的真菌,各验室可以根据自己医院患者真菌感染的特点,建立个性化的数据库,提高真菌检测时效和准确性,更好的指导临床合理用药,有效控制真菌感染。
[Abstract]:Objective fungi, about 100 thousand species of eukaryotes widely distributed in nature, have been discovered, of which about 300 species are pathogenic to humans, the growth characteristics, clinical characteristics and drug resistance of different species of fungi are different. The extensive use of antibiotics and immunosuppressive agents, the increase of chemotherapy and chemotherapy, organ transplantation and interventional technique It leads to an increase in the immune function of the population, causing many opportunistic pathogenic fungi to cause infection and becoming an important cause of death in patients with low immune function. In addition, the patients with liver diseases are susceptible to infection due to low protein, mononuclear macrophage system, leukocyte adhesion chemotactic and phagocytic energy decrease, intestinal flora imbalance and so on. The susceptible population of bacteria infection, the incidence of fungal infection and the mortality rate are increasing year by year, causing high clinical attention. At the same time, with the extensive application of antifungal drugs, the drug resistance of fungi is becoming increasingly prominent, which challenges the clinical experience of drug use. The characteristics of the fungus are slow growth, the traditional culture and identification method takes a long time, 3-7 days, and the growing diversity of fungi, which makes the difficulty of identification more restricts the early diagnosis and targeted treatment of clinical. Matrix assisted laser desorption ionization time of flight mass spectrometry (Matrix-Assisted Laser-Desorption/Ioni zation T) Ime of Flight Mass Spectrometry, MALDI-TOF MS) is a newly developed diagnostic technique in recent years. It can identify bacteria, Mycobacterium, virus and so on by direct detection of biomarkers (proteins). It has a fast, accurate, simple, low cost special point. In this study, the results of identification of fungi by mass spectrometry were compared with biochemical identification, molecular biology and other technical identification results. The method of rapid identification of fungi by mass spectrometry was explored to evaluate the identification ability of fungi and to provide reliable diagnosis for rapid clinical diagnosis of fungal infection. At the same time, the clinical data and drug sensitivity of the fungal infection in the patients with liver disease in our hospital were analyzed, the pathogenic characteristics and the growth trend of the fungal infection of the patients with liver diseases were found, the resistance to different kinds of antifungal drugs was analyzed, and the clinical and rational application of antifungal drugs was better guided to achieve effective control of fungi. Methods the clinical data of 2236 strains of fungi isolated from hospitalized patients with clinical liver disease in 2011 (7 days from the same site of the same patient were separated from the same patient in 7 days), and the characteristics of the susceptible population, the infection site and the distribution of the bacteria group were statistically analyzed. The results were analyzed with fluconazole, amphotericin, itraconazole, and 2236 strains of bacteria. The drug sensitivity test of 2236 strains of fungi was carried out by K-B, and the resistance was analyzed statistically. 327 strains of fungal strains were identified by Brook's Microflex mass spectrometer. The results were compared with the identification results of VITEK-2 (yeast like fungi) and microscopic examination (filamentous fungi), and the differences were identified by molecular biological methods. Results from the analysis of the isolation of fungi from the specimens of liver disease patients in our hospital for the last four years, the fungal infection was mostly in the male patients, the proportion of men and women was 1.73:1, the average age was 54 + 14.79 years. The fungal infection of the liver disease patients was more likely to occur in the decompensated cirrhosis, liver cancer and severe hepatitis patients, and the proportion of the patients with decompensated cirrhosis was 69.65. %, the number of liver cancer patients accounted for the number of 16.97%. fungi from 61 strains in 2011, a sharp increase to 1222 in 2014, up 19.03 times. Four years, 2236 strains of fungi, 1889 yeast like fungi, 84.48%, 347 filamentous fungi, 15.52%. The annual separation rate of yeast like fungi is absolute superiority, 2011 is 63.93%, 2012 is 85.42%, 2013 is 84.. 92%, the species of 85.02%. fungi isolated in 2014 also increased from 15 in 2011 to nearly 30 of the present. Among the various fungal species, the first was Candida albicans (52.64%), followed by Candida tropicalis (13.86%) and Aspergillus fumigatus (12.03%). The fungal infection sites were mostly in the respiratory tract, 66.41%, and 52.24% of the yeast like fungi. Filamentous fungi accounted for 14.18%, and Candida albicans accounted for 35.15%, followed by Aspergillus fumigatus and 11.09%. ascites and drainage is the characteristic of fungal infection in patients with liver disease, the separation rate was 13.91%, the infection species was mainly Candida albicans (8.32%). From our hospital for nearly four years, fungal susceptibility analysis, silk The resistance rate of fungi was generally higher than that of yeast like fungi (P0.05). The resistance rates of yeast like fungi to fluconazole, amphotericin, itraconazole and voriconazole were 11.96%, 2.54%, 1.96% and 0.69%, respectively, and the resistance rates of filamentous fungi to fluconazole, amphotericin, itraconazole and Fu Likang were 95.97%, 17.29%, 6.05% and 4.61%., respectively, 3. The results of mass spectrometric identification of 27 fungi showed that the identification rate of the yeast like fungus score was 90.31% according to the score standard mass spectrometer (2). The identification rate of the 98.68%. filamentous fungi score reached 74%, reaching the level of 1.7, and the level of the genus was 94%. for the common clinical Aspergillus Jian Dingzheng. It is true that the 96.74%. mass spectrometer can also make a more detailed classification of fungi from the species, such as the accurate nearly smooth Candida albicans, similar to Candida smooth and pseudo Candida. The results of the identification of the bacterial protein from the culture bottle directly from the positive culture bottle of the single fungal infection and the mass spectrometry from the culture bottle. The results of identification with bacterial colonies after culture are the same, which can advance the identification time of fungal infection in body fluid 24h.. It is found that it is only 20min for identification of single strains in mass spectrometer, and multiple strains can be detected at the same time. It is the shortest and easy operation method of fungi identification at present. The number and type of fungal infection of the patients with liver disease have been increasing year by year. We should pay special attention to the infection of aseptic fluid such as ascites, drainage fluid and other cases, strengthen the nursing of various indwelling tubes, and strictly aseptic operation for all kinds of interventional therapy. The identification of bacteria in the laboratory and the results of drug sensitivity are reasonable for antifungal treatment, and we should consider the toxicity of the drugs to the liver and choose the antifungal agents carefully. The mass spectrometer has achieved the ideal results on the identification of fungi at the level of species, especially the identification of yeast like fungi and Aspergillus bacteria, which fully meet the clinical microbes. The needs of laboratory fungi detection. The mass spectrometer has a broad clinical application prospect in the diagnosis of fungal infection. For the uncommon fungi, the laboratory can establish a personalized database according to the characteristics of the fungal infection of their own hospital, improve the time and accuracy of fungal detection, guide the clinical rational use of drugs, and effectively control the truth. Bacterial infection.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
本文编号：2171800
[Abstract]:Objective fungi, about 100 thousand species of eukaryotes widely distributed in nature, have been discovered, of which about 300 species are pathogenic to humans, the growth characteristics, clinical characteristics and drug resistance of different species of fungi are different. The extensive use of antibiotics and immunosuppressive agents, the increase of chemotherapy and chemotherapy, organ transplantation and interventional technique It leads to an increase in the immune function of the population, causing many opportunistic pathogenic fungi to cause infection and becoming an important cause of death in patients with low immune function. In addition, the patients with liver diseases are susceptible to infection due to low protein, mononuclear macrophage system, leukocyte adhesion chemotactic and phagocytic energy decrease, intestinal flora imbalance and so on. The susceptible population of bacteria infection, the incidence of fungal infection and the mortality rate are increasing year by year, causing high clinical attention. At the same time, with the extensive application of antifungal drugs, the drug resistance of fungi is becoming increasingly prominent, which challenges the clinical experience of drug use. The characteristics of the fungus are slow growth, the traditional culture and identification method takes a long time, 3-7 days, and the growing diversity of fungi, which makes the difficulty of identification more restricts the early diagnosis and targeted treatment of clinical. Matrix assisted laser desorption ionization time of flight mass spectrometry (Matrix-Assisted Laser-Desorption/Ioni zation T) Ime of Flight Mass Spectrometry, MALDI-TOF MS) is a newly developed diagnostic technique in recent years. It can identify bacteria, Mycobacterium, virus and so on by direct detection of biomarkers (proteins). It has a fast, accurate, simple, low cost special point. In this study, the results of identification of fungi by mass spectrometry were compared with biochemical identification, molecular biology and other technical identification results. The method of rapid identification of fungi by mass spectrometry was explored to evaluate the identification ability of fungi and to provide reliable diagnosis for rapid clinical diagnosis of fungal infection. At the same time, the clinical data and drug sensitivity of the fungal infection in the patients with liver disease in our hospital were analyzed, the pathogenic characteristics and the growth trend of the fungal infection of the patients with liver diseases were found, the resistance to different kinds of antifungal drugs was analyzed, and the clinical and rational application of antifungal drugs was better guided to achieve effective control of fungi. Methods the clinical data of 2236 strains of fungi isolated from hospitalized patients with clinical liver disease in 2011 (7 days from the same site of the same patient were separated from the same patient in 7 days), and the characteristics of the susceptible population, the infection site and the distribution of the bacteria group were statistically analyzed. The results were analyzed with fluconazole, amphotericin, itraconazole, and 2236 strains of bacteria. The drug sensitivity test of 2236 strains of fungi was carried out by K-B, and the resistance was analyzed statistically. 327 strains of fungal strains were identified by Brook's Microflex mass spectrometer. The results were compared with the identification results of VITEK-2 (yeast like fungi) and microscopic examination (filamentous fungi), and the differences were identified by molecular biological methods. Results from the analysis of the isolation of fungi from the specimens of liver disease patients in our hospital for the last four years, the fungal infection was mostly in the male patients, the proportion of men and women was 1.73:1, the average age was 54 + 14.79 years. The fungal infection of the liver disease patients was more likely to occur in the decompensated cirrhosis, liver cancer and severe hepatitis patients, and the proportion of the patients with decompensated cirrhosis was 69.65. %, the number of liver cancer patients accounted for the number of 16.97%. fungi from 61 strains in 2011, a sharp increase to 1222 in 2014, up 19.03 times. Four years, 2236 strains of fungi, 1889 yeast like fungi, 84.48%, 347 filamentous fungi, 15.52%. The annual separation rate of yeast like fungi is absolute superiority, 2011 is 63.93%, 2012 is 85.42%, 2013 is 84.. 92%, the species of 85.02%. fungi isolated in 2014 also increased from 15 in 2011 to nearly 30 of the present. Among the various fungal species, the first was Candida albicans (52.64%), followed by Candida tropicalis (13.86%) and Aspergillus fumigatus (12.03%). The fungal infection sites were mostly in the respiratory tract, 66.41%, and 52.24% of the yeast like fungi. Filamentous fungi accounted for 14.18%, and Candida albicans accounted for 35.15%, followed by Aspergillus fumigatus and 11.09%. ascites and drainage is the characteristic of fungal infection in patients with liver disease, the separation rate was 13.91%, the infection species was mainly Candida albicans (8.32%). From our hospital for nearly four years, fungal susceptibility analysis, silk The resistance rate of fungi was generally higher than that of yeast like fungi (P0.05). The resistance rates of yeast like fungi to fluconazole, amphotericin, itraconazole and voriconazole were 11.96%, 2.54%, 1.96% and 0.69%, respectively, and the resistance rates of filamentous fungi to fluconazole, amphotericin, itraconazole and Fu Likang were 95.97%, 17.29%, 6.05% and 4.61%., respectively, 3. The results of mass spectrometric identification of 27 fungi showed that the identification rate of the yeast like fungus score was 90.31% according to the score standard mass spectrometer (2). The identification rate of the 98.68%. filamentous fungi score reached 74%, reaching the level of 1.7, and the level of the genus was 94%. for the common clinical Aspergillus Jian Dingzheng. It is true that the 96.74%. mass spectrometer can also make a more detailed classification of fungi from the species, such as the accurate nearly smooth Candida albicans, similar to Candida smooth and pseudo Candida. The results of the identification of the bacterial protein from the culture bottle directly from the positive culture bottle of the single fungal infection and the mass spectrometry from the culture bottle. The results of identification with bacterial colonies after culture are the same, which can advance the identification time of fungal infection in body fluid 24h.. It is found that it is only 20min for identification of single strains in mass spectrometer, and multiple strains can be detected at the same time. It is the shortest and easy operation method of fungi identification at present. The number and type of fungal infection of the patients with liver disease have been increasing year by year. We should pay special attention to the infection of aseptic fluid such as ascites, drainage fluid and other cases, strengthen the nursing of various indwelling tubes, and strictly aseptic operation for all kinds of interventional therapy. The identification of bacteria in the laboratory and the results of drug sensitivity are reasonable for antifungal treatment, and we should consider the toxicity of the drugs to the liver and choose the antifungal agents carefully. The mass spectrometer has achieved the ideal results on the identification of fungi at the level of species, especially the identification of yeast like fungi and Aspergillus bacteria, which fully meet the clinical microbes. The needs of laboratory fungi detection. The mass spectrometer has a broad clinical application prospect in the diagnosis of fungal infection. For the uncommon fungi, the laboratory can establish a personalized database according to the characteristics of the fungal infection of their own hospital, improve the time and accuracy of fungal detection, guide the clinical rational use of drugs, and effectively control the truth. Bacterial infection.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5
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1 冯文莉;杨静;奚志琴;朱永乐;张润梅;;3年间医院内侵袭性真菌感染的病原菌分布及临床分析[J];中华医院感染学杂志;2008年07期
2 李慧;;念珠菌420株临床分布及Rosco纸片法药敏结果分析[J];中国误诊学杂志;2011年16期
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