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学科主题: 外科学
题名:
外科感染常见病原菌及耐药基因的可定制化快速检测碟式芯片的开发和应用研究
作者: 韩龙
答辩日期: 2016-05-04
导师: 王杉
专业: 外科学
授予单位: 北京大学
授予地点: 北京大学第二临床医学院
学位: 博士
关键词: 碟式芯片 ; 耐药基因 ; 细菌感染 ; 微流控
其他题名: Development and Application of a Nolve Customized Microfluidic Chip for Rapid Identification of Pathogens and Drug-Resistant Genes based on the Original Bacterial Spectrum of Surgical Infections in the Hospital
分类号: R63
摘要:

背景

感染性疾病仍然是威胁人类健康的主要原因之一,特别是在发展中国家中。据WHO统计,2012年仅下呼吸道感染就在全球造成310万人死亡,占据死亡人数的5.5%。在低收入国家中,下呼吸道感染的死亡率甚至超过心脑血管疾病,达到91/10万人,成为死亡率最高的疾病[1]。外科感染是指需要外科干预的或在围手术期发生的感染,是外科病房常见的疾病,也是外科病人最主要的并发症,其中手术部位感染(surgical site infection, SSI)是排名第二位的住院病人常见并发症。

感染性疾病主要是由细菌引起的,不同的菌种有不同的致病力和毒力。除了外科手术,抗生素治疗也是主要措施之一,因此明确引起感染的病原菌对于感染性疾病的治疗至关重要。目前临床应用抗生素的指征仍然依靠经验性治疗。抗感染治疗可以降低外科感染的死亡率,但是不合理的使用抗生素则使得细菌耐药性不断产生,增加了治疗的困难,影响了患者的生活质量,导致死亡率和并发症增加。

目前传统的细菌检测鉴定方法仍然以培养和生化鉴定为主,检测时间通常为48~72小时,有时甚至1周。虽然培养法被认为是病原菌检测的金标准,但这种方法在临床应用中显示出耗时又费力的缺点,且容易受到取材方法、培养条件的影响。

近年来,分子生物学技术被应用于病原菌的检测中,并以其高特异度、耗时少和交叉感染少的特点赢得关注,其中包括免疫反应、聚合酶链反应(Polymerase Chain Reaction, PCR)检测技术、基因测序、基因芯片等。恒温扩增技术(如环介导恒温扩增(loop-mediated isothermal amplification, LAMP))的出现,使分子检测的技术更加简便、易行,同时灵敏度高、试验条件要求低,具有广阔的发展前景。

微流控芯片以其高效、快速、试验用量少等优点,在氨基酸和蛋白质的分离、免疫分析、DNA 分析和测序、生物细胞研究等方面显示出巨大的潜力,必将对疾病诊断和治疗、新药开发、食品卫生等诸多领域产生革命性的影响。

新型碟式芯片是基于LAMP的方法,在微流控芯片上进行核酸扩增,针对不同的病原菌设计出不同的引物,根据扩增处的片段不同进行诊断。具有快速的特点,最短时间可以达到3小时内,但检测目标数量有限制。

不同医院在不同时段病原菌谱和耐药菌谱上存在差异,在医院内部不同科室和不同标本间常常也会有所不同,选择何种细菌和耐药基因作为检测目标需要根据临床情况不断调整。在进行芯片设计时,需考虑所在单位常见病原菌谱和耐药菌谱,建立筛选模式,覆盖大部分的临床感染病例。

建立基于医院外科感染病原菌谱和耐药菌谱的定制化快速病原菌及耐药基因碟式芯片,实现及时、准确、有效地进行病原学诊断和耐药性评估,改善外科感染的治疗效果,对于减少不合理用药、减少耐药菌的产生和减轻患者的经济负担具有重要的应用价值。

目的

1、根据不同医院在不同时期病原菌谱及耐药菌谱的差异,首先建立相应的定制化病原菌及耐药基因的筛选模式,本研究通过回顾性分析北京大学人民医院医院外科住院感染患者临床病例资料筛选碟式芯片检测靶标。

2、构建基于北京大学人民医院外科感染病原菌谱和耐药菌谱的快速定制化可同时检测病原菌及耐药基因的碟式芯片。

3、评估基于常见病原菌和耐药基因的碟式芯片检测临床标本的诊断效能。建立临床感染病原菌及耐药基因快速可定制化碟式芯片检测平台,为尽早按菌谱和耐药性选择抗菌药物,探索基于医院外科感染病原菌谱和耐药菌谱的快速定制化病原菌及耐药基因检测芯片构建的模式。

材料与方法

1、连续收集北京大学人民医院2012年1月1日~2015年12月31日外科住院病人发生感染的病例资料,共检出来自2644个病人的非重复菌株3617例,通过对临床病例资料、病原菌、药物敏感性试验结果的回顾性分析,建立北京大学人民医院外科感染病原菌谱及耐药菌谱的数据库,分析外科感染病原菌种类、分布及其耐药性的趋势。

2、基于上述结果,建立北京大学人民医院外科感染病原菌谱和耐药菌谱的筛选模型。

3、通过生物信息学数据库GenBank、PubMed检索病原菌及耐药基因全基因序列,通过生物学分析软件BioEdit和AlignX进行每个基因的序列间两两比对分析,使用软件Primer Explorer V4设计病原菌及耐药基因引物。

4、利用芯片检测体系构建方法,构建基于医院外科感染病原菌谱和耐药菌谱的快速定制化病原菌和耐药基因碟式芯片。

5、应用定制化外科感染病原菌和耐药基因碟式芯片对2015年7月24日至2015年12月1日收集的189份不同类型感染标本(脓、痰、引流液、伤口分泌物等)进行检测,结合常规培养鉴定和药敏试验、基因测序方法验证和评价碟式芯片的诊断效能。

结果

共收集北京大学人民医院外科感染来自2644个病人的非重复病原菌3617株,其中主要以革兰氏阴性菌感染为主,占60.4%(包括大肠埃希菌20.35%、肺炎克雷伯菌8.99%和铜绿假单胞菌5.97%);革兰氏阳性球菌占39.6%(包括凝固酶阴性葡萄球菌16.86%、肠球菌10.01%及金黄色葡萄球菌5.31%),前十位病原菌为大肠埃希菌、凝固酶阴性葡萄球菌、肺炎克雷伯菌、铜绿假单胞菌、粪肠球菌、金黄色葡萄球菌、鲍曼不动杆菌、屎肠球菌、奇异变形杆菌和阴沟肠杆菌。

不同时间外科感染病原菌谱构成不同。基于医院4年外科病原菌谱分析,共培养出细菌135种,每年培养出的细菌种类各不相同,分别为69种、75种、79种、84种,其中每年均能检测出的细菌共38种,四年中仅能检测出1株的细菌共43种。大肠埃希菌和凝固酶阴性的葡萄球菌一直是外科感染中占比例最大的菌种,但是趋势则表现不同。大肠埃希菌保持下降趋势;凝固酶阴性葡萄球菌则呈现上升的趋势。铜绿假单胞菌保持下降趋势,已经从2012年的6.34%,降低至2015年的5.71%。肺炎克雷伯菌初期维持上升趋势,至2013年开始恒定在9%左右。其他菌种无明显变化或无规律变化。不同时间中,前十位病原菌种类基本一致。

不同外科亚专科主要病原菌构成不同。骨科常见前三位病原菌为凝固酶阴性葡萄球菌(25.35%)、大肠埃希菌(18.41%)、金黄色葡萄球菌(10.20%);普外科常见前三位病原菌依次为大肠埃希菌(21.06%)、肺炎克雷伯菌(12.27%)、凝固酶阴性葡萄球菌(11.94%);泌尿外科常见前三位病原菌为大肠埃希菌(27.70%)、凝固酶阴性葡萄球菌(17.06%)、粪肠球菌(10.56%);神经外科常见前三位病原菌为鲍曼不动杆菌(18.52%)、肺炎克雷伯菌(14.81%)、凝固酶阴性葡萄球菌(10.19%);心胸外科常见前三位病原菌为凝固酶阴性葡萄球菌(17.92%)、肺炎克雷伯菌(17.19%)、铜绿假单胞菌(11.14%)。各亚专科病原菌构成组间比较差异有显著性统计学意义(P<0.05),前十位菌种基本保持恒定。

不同标本类型组要病原菌不同。尿液标本中共培养出70种细菌,主要为大肠埃希菌(30.78%)、凝固酶阴性葡萄球菌(15.70%)和粪肠杆菌(11.55%);痰液标本中共培养出43种细菌,主要为肺炎克雷伯菌(22.17%)、鲍曼不动杆菌(18.93%)和铜绿假单胞菌(14.24%);血液中共培养出59种细菌,主要为凝固酶阴性葡萄球菌(24.75%)、大肠埃希菌(24.50%)和肺炎克雷伯菌(8.42%);伤口分泌物中共培养出43种细菌,主要为大肠埃希菌(21.07%)、凝固酶阴性葡萄球菌(18.99%)和金黄色葡萄球菌(15.73%)。不同临床标本病原菌构成有显著性差异(P<0.05),前十位保持恒定。

筛选出占比最多的前十位细菌,分别是大肠埃希菌、肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌、阴沟肠杆菌、金黄色葡萄球菌、凝固酶阴性葡萄球菌、粪肠球菌、屎肠球菌和奇异变形杆菌,作为芯片构建的靶标。

针对外科病原菌耐药性的分析结果显示,革兰阳性菌对青霉素类、大环内酯类和林克酰胺类抗生素的耐药率较高,革兰阴性菌对青霉素类、头孢菌素类、碳青霉烯类、氨基糖苷类和喹诺酮类抗生素的耐药性较高。在设计芯片时,需根据临床常用抗生素对常见病原菌的耐药性来选择相应的耐药基因,指导临床用药。

在多重耐药菌的分析中,产ESBLs的大肠埃希菌的比例约为63.05%~69.59%,且逐年缓慢下降;肺炎克雷伯菌中,产ESBLs菌株的比例为21.98%~34.09%,逐年呈现下降趋势;金黄色葡萄球菌中,MRSA的检出比例在2012年为33.33%,2013年降至25.00%,但从2013年开始逐步升至2015年的43.10%;凝固酶阴性的葡萄球菌中,MRCNS变化不明显。

通过耐药数据分析及耐药菌流行病学文献筛选出OXA23、OXA24、OXA58、OXA66、CTX-M1、CTX-M9、DHA-1、VEB、GES-1、ACT-1、CMY-2、KPC、IMP-4、VIM、NDM1、aacC1、aadA1、mecA、ermB、mefA、mefE、vanA、vanB共23种耐药基因为本研究耐药芯片检测目标基因。

针对10种病原菌和23个耐药基因,在Genbank中检索病原菌及耐药基因DNA序列,建立病原菌和耐药基因序列数据库。经参考品菌株验证优化设计引物,保证高灵敏度和特异度。

在同一张芯片构建基于北京大学人民医院外科感染病原菌谱和耐药菌谱的快速定制化病原菌及耐药基因碟式芯片。

应用外科感染病原菌和耐药基因碟式芯片可在同一芯片同时检测病原菌及耐药基因;菌种鉴定灵敏度70.15%(95%CI, 57.57%-80.40%),特异度为94.80%(95%CI, 89.14%-96.01%),对临床标本中耐药菌耐药基因检测特异性为94.44~100%。

碟式芯片检测临床样本时间2.68±0.39h,传统方法检测时间为71.02±30.01h。

结论

1、基于不同医院外科感染病原菌谱及耐药菌谱构建快速定制化碟式芯片模式可成为定制化芯片构建和应用的新模式。

2、碟式芯片可用于外科感染病原菌和耐药基因的检测,可以在3小时内完成不同类型临床样本的检测,但其灵敏度尚需要进一步提高。

3、基于医院外科感染病原菌谱和耐药菌谱构建快速定制化病原菌和耐药基因检测碟式芯片的模式可为探索及时、准确、有效的临床抗感染治疗、提高外科感染治疗效果、促进抗菌药物的合理使用提供新的方法。

英文摘要:

Background

Infectious diseases are still main threat of human health in the world, especially in developing countries. According to WHO, there were 3.1 million deaths due to lower respiratory tract infection in 2012, that was 5.5% of all deaths worldwide. The mortality of lower raspiratory tract infections, which account for 91 death per 1 hundred thousand population, is the highest in low-income countries, even higher than that of the cardiovascular diseases[1]. Surgical infection refers to infecions that is in the need for surgical treatment or occur during the perioperative period. It’s not only the most common disease in the surgical ward, but also the most common complications in the perioperative period, among which surgical site infections (SSI) are the second common complications during hospital stay.

Infections are mainly caused by bacteriums of different virulence and toxicity. In addition to surgical treatment, the application of antibacterial drugs is still an indispensable means for treatment, which makes it a priority to identify the causing pathogens as soon as possible. Clinically antibacterial treatment is still carried out based on experience. In general, antibacterial drugs could reduce the the mortality rate of patients with surgical infection, but the irrational use of antibacterial drugs has led to a rapid increase in drug resistants, which makes it difficult when choosing the susceptible antibacterial drug and affect the quality of life of patients, and ultimately result in the increase of morbidity and mortality.

The conventional pathogen identification methods are still based on culture and biochemical identification, a process typically takes 48 to 72 hours, or even one week. Although considered the “golden standard”, conventional culture based methods share the disadvantage of time consuming, laborious work and vunerable to sampling methods and culture conditions.

The effert in the development of molecular microbiology for identification of pathogenic organisms provides faster, easier and more accurate diagnostic methods, including immunological method, polymerase chain reaction (PCR), gene sequencing and DNA chip. Isothermal amplification methods (for example loop-mediated isothermal amplification, LAMP) provide more easy-to-use, high sensitivity and less device dependent methods, expecting more clinical use in this future.

Microfluidic chip has the advantage of rapid detection, high effectiveness and less reagent consuming, which can be used in the immunological analysis, DNA sequencing and cell research. It will change the way we draw a diagnose decision, and will have a great influence in the development of new drugs and public health.

A novel disk chip based upon LAMP has been developed. Different genes can be amplificated in the reaction chamber using different primers and come to a diagnosis of the genes within the samples. The prosess can be done in less than 3 hours but with limited targets.

There are differences in the microbiology spectrum and antibacterial resistance in different hospitals and in different times. There are also differences between different specimens and different departments. The target genes of the disk chip should been carefully chosen and d according to the clinical requirement. In order to do that, we reviewed the most common pathogens and antibacterial resistance in the hospitals, then set up a model for target filtering.

Disk chip technology can compensate for the deficiencies of the conventional detection, it achieve parallel processing of multiple targets, using fast, accurate and high-sensitivity detection method. Therefore, the establishment of rapid disk chip technology platform for rapid, accurate and effective clinical identification of pathogens to improve the prognosis of patients with surgical infections, will play an important role in the reduction of irrational antibacterial use, hospitalization costs and the emergence of antibacterial resistance.

Objective

In different hospitals and at different times, there are different spectrums of pathogens and antibacterial resistance, so a pathogenic bacterium and antibacterial resistance filter mode is needed before the development of a novel microfluidic chip. This study is a retrospective analysis of Peking University People’s Hospital surgical infections in hospitalized patients, in order to sellect the most common pathogens and resistance genes we need to identify in the next step.

Build a customized novel microfluidic chip based on the bacterial spectrum and antibacterial resistance spectrum in the surgical ward that can simultaneously detect pathogens and antibacterial resistant genes.

Primary application of microfluidic chip to identify clinical samples of surgical infection to assess the diagnositic efficency. The establishment of the platform which can identify clinical surgical infection pathogens and antibacterial resistance genes in a short time enables ion of antibiotics based on indication as soon as possible and can be used as a hospital model for hospital customized rapid detection of pathogenic bacterium and resistance genes.

Methods

Collect data of surgical infection in Peking University People's Hospital from January 1 2012 to December 31 2015. A total of 3617 non-duplicate bacterias have been identified from 2644 patients in the surgical ward. Retrospective analysis of the distribution of bacteria and the results of susceptibility test was made to establish surgical infection bacterial spectrum and antibacterial-resistant spectrum database to get a better understanding of surgical infection pathogen distributions and trends of antimicrobial resistance.

Based on the result above, a filter mode to target pathogens and resistance genes was establishied.

Search for gene sequences of ed pathogens and resistance genes from bioinformatics database (GenBank, PubMed). Compare gene sequences of the ed targets by analytic software BioEdit and AlignX. The design of the primers for LAMP of the ed targets was made by Primer Explorer V4.

The microfluidic chip construction method was used to build a novel rapid microfluidic chip of pathogens and resistance genes for surgical infectionsy.

The novel rapid microfluidic chip of pathogens and resistance genes for surgical infectionsy was used to identify 189 clinical specimens (including absess, sputum, drainage and wound), compared with conventional culture-based identification and gene sequencing to evaluate the efficiency.

Result

A total of 3617 pathogens from 2645 patients were analysed mainly account for gram-negative bacterias (60.4%, including Escherichia coli 20.35%, Klebsiella pneumoniae 8.99% and Pseudomonas aeruginosa 5.97%); gram-positive bacterias account for 39.6 % (including coagulase-negative Staphylococci 16.86%, enterococci 10.01% and Staphylococcus aureus 5.31%) in surgical ward. The top ten pathogens were Escherichia coli, coagulase-negative Staphylococci, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Acinetobacter baumannii, Enterococcus faecium, Enterobacter cloacae and Proteus mirabilis.

The proportions of pathogens in different periods were different. A total of 135 kinds of bacterias were found in the specimens from patients with surgical infecitons, in each year 69, 75, 79 and 84. Among them 38 kinds of bacterias can be found every year and 43 can only be found once in 4 years. Escherichia coli and coagulase negative Staphylococcus are the biggest portions with different trends. Escherichia coli keeps decreasing and coagulase negative Staphylococcus is increasing. Pseudomonas aeruginosa decreased slowly in four years from 6.34% in 2012 to 5.71 in 2015. Klebsiella pneumoniae increased slightly and the were stable at 9%. The other bacterias have not change significantly or changes irregularly. In different periods, the top ten kinds of bacterias are the same.

The proportions of pathogens in different surgical departments were different, but they was basically the same pathogens. In orthopedic department, top three pathogens were coagulase negative staphylococci (25.35%), Escherichia coli (18.41%), Staphylococcus aureus (10.20%). In general surgery department, top three pathogens were Escherichia coli (21.06%), Klebsiella pneumoniae (12.27%), coagulase negative Staphylococci (11.94%). In urology department, top three pathogens were Escherichia coli (27.70%), coagulase negative Staphylococci (17.06%), Enterococcus faecalis (10.56%). In cardiothoracic department, top three pathogens were coagulase negative Staphylococci (17.92%), Klebsiella pneumoniae (17.19%) and Pseudomonas aeruginosa (11.14%). The proportions of pathogens in different surgical departments were different (p <0.05), top ten types of pathogens are the same.

The proportions of pathogens in different specimen types were different. In the urine specimens most common pathogens were Escherichia coli (30.78%), coagulase negative Staphylococci (15.70% ) and Enterococcus faecalis (11.55%). In sputum, Klebsiella pneumoniae (22.17%), Acinetobacter acinetobacter (18.63%) and Pseudomonas aeruginosa (14.24%). In blood samples coagulase negative Staphylococci (24.75%), Escherichia coli (24.50%) and Klebsiella pneumoniae (8.42%). In drainage specimens Escherichia coli (21.07%), coagulase negative Staphylococci (18.99%) and Staphylococcus aureus (15.73%). Constitutes of pathogen in different clinical specimens were significantly different (p <0.05), top ten types of pathogens are the same.

The most common pathogens, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Staphylococcus aureus, coagulase negative Staphylococci, Enterococcus faecalis and Enterococcus feces were ed as the targets of the microfluidic chip.

The analysis of antibacterial resistance in the surgical ware shows, Gram positive bacterias were commonly resistant to antibacterial of penicillin, macrolides and lincosaes while Gram negative bacterias to antibacterial of penicillins and cephalosporins, carbapenems, aminoglycosides and quinolones.

The production of extended-spectrum beta-lactamase (ESBL) Escherichia coli proportion decreased from 69.59% to 63.05%. ESBL-producing Klebsiella pneumoniae proportion decreased from 34.09% to 21.98%. Methicillin-resistant Staphylococcus aureus proportion increased from 25.00% in 2013 to 43.10% in 2015. Methicillin-resistant coagulase negative Staphylococcus did not change much.

By analysis of resistant pathogens data and review of literature, a total of 23 resistant genes were ed as the targets including OXA23、OXA24、OXA58、OXA66、CTX-M1、CTX-M9、DHA-1、VEB、GES-1、ACT-1、CMY-2、KPC、IMP-4、VIM、NDM1、aacC1、aadA1、mecA、ermB、mefA、mefE、vanA、vanB.

Search for 10 pathogens and 23 resistant genes in GenBank to retrieve DNA sequences of pathogens and resistant genes, establish pathogenic bacteria and drug resistance gene sequence databases.

Successfully invented a novel rapid microfluidic chip based on the surgical infection pathogens and resistant genes of Peking University People's Hospital.

The surgical infection pathogens and resistant genes can be detected in the same chip simultaneously with a sensitivity of 70.15% (95%CI, 57.57%-80.40%) and specificity of 94.80% (95%CI, 89.14%-96.01%).

The novel microfluidic chip can get result in 2.68 ± 0.39h, while traditional methods 71.02 ± 30.01h.

Conclusion

Based on different hospital surgical infection pathogens spectrum and antibacterial resistant, the construction of customized rapid diagnostic chip can be a new model to develop a novel microfluidic chip.

The chip could be used in the detection of pathogens and resistant genes within 3 hours in the variety of specimen types but need to improve the sensibility.

Based on hospital surgical infection pathogen spectrum and resistant genes, the building of customized rapid pathogen and resistant gene detection chip can be a new therapeutic path for rapid, accurate and effective clinical antibacterial treatment and improve the prognosis of surgical infection and promote the rational use of antibacterial agents.

语种: 中文
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内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/124607
Appears in Collections:北京大学第二临床医学院_学位论文

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作者单位: 北京大学第二临床医学院

Recommended Citation:
韩龙. 外科感染常见病原菌及耐药基因的可定制化快速检测碟式芯片的开发和应用研究[D]. 北京大学第二临床医学院. 北京大学. 2016.
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