肝病患者真菌的感染特點(diǎn)和質(zhì)譜快速鑒定研究
發(fā)布時(shí)間：2018-08-08 12:34
【摘要】：目的真菌是廣泛分布于自然界的真核生物,已被發(fā)現(xiàn)的約10萬種,其中對(duì)人類具有致病性約300種,不同種真菌的生長(zhǎng)特性、臨床特點(diǎn)及耐藥性不同。由于抗菌藥物和免疫抑制劑的廣泛應(yīng)用、放化療治療的增加、器官移植及介入技術(shù)操作的開展,導(dǎo)致機(jī)體免疫功能低下人群增加,使很多機(jī)會(huì)致病真菌引起感染,成為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、單核巨噬細(xì)胞系統(tǒng)受損、白細(xì)胞粘附趨化和吞噬能力降低、腸道內(nèi)菌群失調(diào)等原因成為易感染真菌感染的易感人群,真菌感染的發(fā)病率和病死率呈逐年上升趨勢(shì),引起臨床的高度關(guān)注。同時(shí),隨著抗真菌藥物的大量應(yīng)用,真菌的耐藥性也日益突出,為臨床的經(jīng)驗(yàn)用藥提出了挑戰(zhàn)。快速明確感染真菌的特點(diǎn)及耐藥狀況,成為臨床救治的關(guān)鍵。真菌的特點(diǎn)是生長(zhǎng)緩慢,傳統(tǒng)的培養(yǎng)鑒定方法所需時(shí)間較長(zhǎng),一般3-7天,且日益多樣化的真菌,使鑒定難度不斷增加均限制了臨床的早期診斷和針對(duì)性治療�；|(zhì)輔助激光解吸電離飛行時(shí)間質(zhì)譜(Matrix-Assisted Laser-Desorption/Ioni zation Time of Flight Mass Spectrometry,MALDI-TOF MS)是近年快速發(fā)展起來的一種新興的診斷技術(shù),可以通過直接檢測(cè)生物標(biāo)志物(蛋白)來鑒定細(xì)菌、分枝桿菌、病毒等,具有快速、準(zhǔn)確、簡(jiǎn)便、成本低的特點(diǎn)。但它對(duì)真菌的鑒定評(píng)價(jià)說法不一,主要原因真菌的細(xì)胞壁厚,前處理方法不成熟導(dǎo)致鑒定結(jié)果的差異。為此本研究通過把質(zhì)譜儀鑒定真菌的結(jié)果與生化鑒定、分子生物學(xué)等技術(shù)鑒定結(jié)果比對(duì),探索質(zhì)譜技術(shù)快速鑒定真菌的方法,評(píng)價(jià)其真菌鑒定能力,為臨床快速診斷真菌感染提供可靠的依據(jù);同時(shí)對(duì)我院肝病患者真菌感染的臨床資料和藥敏情況進(jìn)行統(tǒng)計(jì)分析,找到肝病患者真菌感染的病原組成特點(diǎn)及增長(zhǎng)趨勢(shì),分析其對(duì)不同種類抗真菌藥物的耐藥性,更好的指導(dǎo)臨床合理的應(yīng)用抗真菌藥物治療,達(dá)到有效控制真菌感染的目的。方法收集我院2011年-2014年分離自臨床肝病住院患者的2236株真菌的臨床資料(7天內(nèi)分離自同一患者相同部位的真菌不重復(fù)統(tǒng)計(jì)),對(duì)其易感人群、感染部位及菌群分布等特點(diǎn)進(jìn)行統(tǒng)計(jì)分析。用氟康唑、兩性霉素B、伊曲康唑、伏立康唑?qū)?236株真菌進(jìn)行K-B法藥敏試驗(yàn),統(tǒng)計(jì)分析其耐藥性。用布魯克公司的Microflex型質(zhì)譜儀對(duì)其中327株真菌菌株進(jìn)行快速鑒定,結(jié)果與VITEK-2(酵母樣真菌)和顯微鏡檢查(絲狀真菌)的鑒定結(jié)果比對(duì),有差異的用分子生物學(xué)方法確認(rèn)鑒定。結(jié)果從我院近四年肝病患者標(biāo)本分離真菌的情況分析,真菌感染多發(fā)于男性患者,男女比例為1.73:1,平均年齡為54±14.79歲。肝病患者真菌感染更易發(fā)生在肝硬化失代償期、肝癌及重癥肝炎患者,其中肝硬化失代償期的患者所占比例為69.65%,肝癌患者占16.97%。真菌的分離數(shù)量從2011年的61株,急劇增長(zhǎng)到2014年的1222株,增長(zhǎng)了19.03倍。四年共分離真菌2236株,酵母樣真菌1889株,占84.48%,絲狀真菌347株,占15.52%;酵母樣真菌的年分離率占絕對(duì)優(yōu)勢(shì),2011年為63.93%,2012年為85.42%,2013年為84.92%,2014年為85.02%。真菌分離的種類也由2011年的15種增加到了目前的近30種。在眾多真菌種類中,居首的為白假絲酵母菌(52.64%),其次為熱帶假絲酵母菌(13.86%)和煙曲霉菌(12.03%)。真菌感染部位多發(fā)于呼吸道,占66.41%,其中酵母樣真菌占52.24%,絲狀真菌占14.18%;呼吸道分離的真菌種類以白假絲酵母菌為主,占35.15%,其次為煙曲霉菌占11.09%。腹水和引流液感染是肝病患者真菌感染的特點(diǎn),分離率為13.91%,感染種類以白假絲酵母菌為主(8.32%)。從我院近四年的真菌藥敏結(jié)果分析,絲狀真菌的耐藥率普遍高于酵母樣真菌(p0.05),酵母樣真菌對(duì)氟康唑、兩性霉素、伊曲康唑和伏立康唑的耐藥率分別為11.96%,2.54%,1.96%和0.69%;絲狀真菌對(duì)氟康唑、兩性霉素、伊曲康唑和伏立康唑的耐藥率分別為95.97%,17.29%,6.05%和4.61%。本研究對(duì)327株真菌的質(zhì)譜鑒定結(jié)果顯示,依照其評(píng)分標(biāo)準(zhǔn)質(zhì)譜儀鑒定酵母樣真菌評(píng)分達(dá)到種水平(評(píng)分2.0)的鑒定率為90.31%,達(dá)到屬水平(評(píng)分1.7)的為98.68%。絲狀真菌評(píng)分達(dá)到種水平的鑒定率為74%,達(dá)到屬水平的為94%。對(duì)臨床常見的曲霉菌的鑒定正確率可以達(dá)到96.74%。質(zhì)譜儀還能從種內(nèi)對(duì)真菌進(jìn)行更細(xì)致的分型,例如可準(zhǔn)確的將近平滑假絲酵母菌,似平滑假絲酵母菌和擬平滑假絲酵母進(jìn)行區(qū)分。對(duì)單一種類真菌感染的陽性培養(yǎng)瓶,質(zhì)譜從培養(yǎng)瓶中直接提取菌種蛋白鑒定的結(jié)果與培養(yǎng)后用菌落鑒定的結(jié)果相同,這可以把體液真菌感染的鑒定時(shí)間提前24h。本研究通過對(duì)現(xiàn)有各種真菌鑒定方法用時(shí)的比較,發(fā)現(xiàn)質(zhì)譜儀單株菌鑒定用時(shí)僅需20min,并且可同時(shí)檢測(cè)多株菌,是目前真菌鑒定用時(shí)最短,操作也較簡(jiǎn)便的方法,為臨床真菌感染的診斷和治療爭(zhēng)取了時(shí)間。結(jié)論肝病患者真菌感染的數(shù)量和種類有逐年上升的趨勢(shì),臨床要特別重視此類患者腹水、引流液等無菌體液的感染,加強(qiáng)對(duì)各種留置管的護(hù)理,對(duì)各種介入性治療要嚴(yán)格無菌操作。應(yīng)根據(jù)微生物實(shí)驗(yàn)室的菌種鑒定和藥敏結(jié)果合理進(jìn)行抗真菌治療,并且要考慮到藥物對(duì)肝臟的毒性,慎重選擇抗真菌藥物。質(zhì)譜儀在鑒定真菌的種屬水平上都達(dá)到了理想的結(jié)果,尤其鑒定酵母樣真菌和曲霉菌的能力更為突出,完全滿足臨床微生物實(shí)驗(yàn)室真菌檢測(cè)的需求。質(zhì)譜儀在真菌感染診斷方面有廣闊的臨床應(yīng)用前景,對(duì)于不常見的真菌,各驗(yàn)室可以根據(jù)自己醫(yī)院患者真菌感染的特點(diǎn),建立個(gè)性化的數(shù)據(jù)庫,提高真菌檢測(cè)時(shí)效和準(zhǔn)確性,更好的指導(dǎo)臨床合理用藥,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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.5
本文編號(hào)：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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R446.5
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2 李慧;;念珠菌420株臨床分布及Rosco紙片法藥敏結(jié)果分析[J];中國誤診學(xué)雜志;2011年16期
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