【作者】 刘春晓；

【导师】 聂军；

【作者基本信息】 南方医科大学 ， 流行病与卫生统计学， 2013， 博士

【摘要】 背景蚊传疾病(mosquito-borne diseases)在世界范围内的威胁日益严重。例如原本流行在非洲的西尼罗热,1999年-2012年在美国爆发流行；登革热、基孔肯雅热的流行区域也在不断扩大。WHO估计,每年感染登革病毒的人群可能达到1亿人。我国广东省每年都有登革热的流行,2010年我国东莞地区还爆发了输入性基孔肯雅热疫情。2008年全国各口岸共发现输入性传染病病例136例,其中输入性登革热病例85例,输入性基孔肯雅热病例5例,占输入性传染病病例总数的66%。大部分蚊传疾病,没有可靠的疫苗,也没有特异性治疗手段。蚊媒监测和控制就成为这些传染病防制的主要手段。蚊媒监测一方面可以了解媒介蚊虫的种群构成、密度大小、季节消长等；另一方面,还可以对蚊传疾病的流行进行预警。因此,国境口岸高度重视蚊媒监测工作,对口岸区域、出入境交通工具等进行了系统的监测。蚊虫传播传染病的能力有种属特异性。不同蚊种传播疾病的效能相差极大,有的不传播疾病,有的高度危险。因此,对蚊虫进行准确的分类鉴定,是蚊传疾病防制的基础。传统的形态学分类依赖鉴定者的主观判断,需要丰富的实践经验,往往需要十几年甚至数十年的积累才能成就一个形态学鉴定专家。大部分实验室缺乏经验丰富的形态学鉴定专家。目前蚊虫分类鉴定主要参考资料是工具书和检索表,但工具书使用的都是模式图,与镜下实际观测视觉差距较大。一些近似种和复合种团,形态差距细微,鉴定就更为困难,单纯依赖形态特点鉴定常常出现争议。口岸实验室对蚊虫分类鉴定的能力参差不齐。2005-2007年,全国口岸共捕获输入性蚊虫28.8万余只,针对部分港口的抽样调查发现,入境船舶蚊虫携带率高达18%-48%。但是这些上报数据中,有三分之一的蚊虫没有分类到种。目前,口岸蚊虫分类鉴定工作存在的问题主要有：专业人才缺乏、鉴定参考资料单一、鉴别能力参差不齐、监测数据没有包括关键的地理和生态信息、数据缺乏共享机制等；更重要的是,尚未建立一个质量控制体系来评估全国各口岸实验室分类鉴定的准确性。这些问题,在疾控系统各级病媒生物实验室也或多或少存在。研究目的1、建立高效的口岸蚊类辅助鉴定方法。研究开发图文并茂的电子化自助检索鉴定系统,改变目前鉴定参考资料单一的局面。2、探索建立基于基因的蚊类鉴定方法。研究从遗传本质上,基于基因对蚊虫进行分类鉴定的方法,提供基因鉴定检索。3、建立口岸蚊类信息化数据平台。通过平台实现数据共享,并能对监测数据自动化、智能化分析和预警,为决策提供支援。4、建立口岸蚊虫分类鉴定实验室质量控制体系。从实验室内部、实验室间两个角度对口岸实验室蚊虫分类鉴定工作进行质量控制。研究方法1、建立常见蚊类形态学数据库采集蚊幼虫,在实验室饲养、羽化,获得鉴定细节完整无缺的标本,经形态学权威专家分类鉴定,制作标准化实物标本。对实物标本的每一个鉴定细节进行高分辨图像采集,获得细节图片,并辅以详尽文字介绍,制作电子化标本。在低倍镜下对整只蚊标本进行多层级拍照,利用multi-focus image fusion技术进行多焦面图像合成,获得兼顾景深和清晰度的整体图片。利用DHTML技术对整体图片和细节图片进行准三维合成,制作常见蚊种的数字化标本。2、建立常见蚊类COI基因数据库探索利用蚊虫线粒体细胞色素氧化酶第一亚基(COI)基因序列鉴定我国常见蚊种的可行性。采集常见蚊种,利用通用引物,扩增COI基因序列,并测序。采用Biodeit软件对序列进行校正,利用MEGA5.0软件进行基因同源性比较和系统进化树分析。探讨COI基因作为我国常见蚊种分类依据的可行性。通过采集蚊种自行测定、基因条形码数据库(BOLD)匕对分析下载等方法,建立常见蚊种COI基因数据库。3、建立口岸蚊类信息化数据平台利用制作的电子化和数字化标本,借助Oracle数据库,基于J2EE技术搭建口岸蚊类信息化数据平台,研发多种形式的辅助鉴定方法。完善监测数据的地理信息、生态信息等,并对数据进行共享。利用GIS技术原理,设计分析模块,实现监测数据自动化、智能化分析。综合各监测点蚊类监测数据,以及相应的地理信息、生态信息等,利用空间分析、时间分析等方法进行预警。4、建立蚊虫分类鉴定实验室质控体系利用蚊虫的标准化实物标本作为鉴定工作的实物参考标准。利用电子化、数字化标本作为鉴定工作的数码参考标准。建立基于ISO:IEC17025的蚊虫鉴定实验室质量管理体系。利用制作的标准化实物标本,开展成蚊形态学鉴定实验室能力验证活动。研究结果1、常见蚊类形态学数据库制作了标准化实物标本11属59种,共2000余只蚊虫标本,涵盖了国境口岸常见的种类。蚊虫标本均由实验室饲养羽化的方法获得,并经权威形态学专家双人鉴定。制作了5属41种蚊的电子化标本,占国境口岸已发现种类的51%,涵盖了常见蚊种(种群构成超过1%)。电子化标本包括：每一个鉴别细节高清晰数码照片、相对应的准确详尽的文字描述；蚊种的生态习性、与传染病关系等资料。数字化标本的种类也是5属41种蚊虫。每种蚊虫建立一张准三维图片,整体视觉上与体视镜低倍视野下肉眼观测的图片无差异。点击任意鉴别细节,都可实现局部放大,出现该细节的高清晰实拍图。这是通过图像合成技术,调用预先在高倍镜下拍摄的图片实现的。2、常见蚊类COI基因数据库共测得常见14种40株成蚊COI基因序列,蚊虫采集自云南普洱、广东广州、广东深圳三地。序列分析表明,各蚊种COI基因扩增片断稳定,各株序列均无缺失,核苷酸长度为415bp。序列的A+T碱基含量范围为69.1%-71.5%,同蚊种不同地理株之间同源性介于97.5%-100%,不同种之间的同源性介于83.5%-93.7%。种内遗传距离介于0-2.5%,种间遗传距离介于6.6%-18.8%。基因聚类分析发现,同种不同地理株首先聚为一类,其次各属蚊种分别聚类,如库蚊属、伊蚊属、阿蚊属,再后是库蚊亚科的三个属聚类,按蚊亚科则聚类为不同的分支,和形态学分类相一致。对复合种团的近似蚊种也可以有效区分。表明使用该段序列对常见蚊虫分类可行。通过采集蚊种自行测定、基因条形码数据库(BOLD)比对分析下载等方法,建立了常见36种蚊种的基因化数据库,包括了复合种团。数据库内容包括核苷酸序列、实验方法、参考文献等部分。使用者可以根据数据库提供的方法进行序列测定,也可以查阅附后的参考文献进行测定。3、信息化数据库平台的建立利用数据库资源为核心,建立基于检验检疫系统内部网络的信息化数据平台,使得各口岸实验室可以方便访问。该平台提供4种辅助鉴定模式：①层级分类检索。采用二叉树式设计,结构层级来自我国蚊种检索表,每一个鉴定细节都配有实拍的高清晰图片和电子化介绍,与镜下观测的实物之间无差异,检测人员可以按照层级,逐步完成分类鉴定。②图片比对检索。选定数据库种中蚊虫鉴定细节图片,直接与待测蚊对比,快速缩小范围,进而确定具体种类。③特征检索。根据提示选定待测蚊的几个鉴别细节特征,在数据库中进行检索。通过检索定位到某些种、属,一一对比相应的标本图片及描述,完成鉴定。④基因检索。输入待测蚊种COI基因的全部或部分序列,系统自动比对,根据同源性从高到低,给出最相近3个种,并提供同源性比率。信息化数据库平台具有监测数据录入、监测点的地理和生态信息收集、数据分析、质量控制等功能；可对监测鉴定数据进行自动化、智能化分析,生成相应的分析图表,还可以结合地理信息、生态信息、监测数据进行空间预警,提供决策支援。利用信息化数据平台的分析思路,我们分析了广东省2001-2006年登革热疫情空间分布模式,表明广州市周边及潮汕地区是广东省登革热流行高发区,与其后的流行数据一致。信息化数据平台的辅助鉴定、数据分析等设计思路,得到了WHO专家的高度认可,将在此基础上搭建全球蚊类监测信息平台。WHO与国家质检总局已经签署初步合作协议。4、数据库在实验室质控的应用利用建立的实物标本、电子化和数字化标本作为实验室鉴定质控溯源依据,参照ISO:IE17025体系,在深圳国检局医学媒介生物实验室建立了质量控制体系,并通过了CNAS的评审,成为全国首家通过CNAS认可的同类实验室。利用制作的蚊虫标准化实物标本,作为盲样,对全国的同类实验室开展了“成蚊形态学鉴定能力验证”工作,这是我国首次开展该项工作。共有28家实验室参加,一次通过率为：85.7%。三种目标蚊种：中华按蚊、致倦库蚊、白纹伊蚊鉴定总准确率为96.2%。四种干扰蚊种：骚扰阿蚊、埃及伊蚊、三带喙库蚊、微小按蚊鉴定总准确率：81.2%,其中微小按蚊鉴定准确率最低,为68.5%。对口岸部分实验室分发标准化标本,作为实验室鉴定工作的实物参考标准。信息化数据平台中的电子化标本、数字化蚊标本,作为口岸实验室鉴定工作的数码参考标准。数字化准三维标本还可作为盲样进行实验室在线鉴定比对,是实验室间质量控制的新手段。结论本研究为蚊媒准确鉴定及监测数据分析、实验室质控提供了系统的解决思路。以自行建立的蚊类鉴定相关数据库作为核心资源,搭建了“口岸蚊类信息化数据平台”。平台形成了一套图文并茂的常见蚊类鉴定指导资料,提供四种辅助鉴定模式,改变了目前单一的“检索表+模式图”的鉴定方式。该平台数据还可作为培训教材,提高各实验室鉴定工作的技术水平。更重要的是,数据库的图片均来自标准化标本,可以作为参考标准,对日常检测鉴定进行质量控制。通过实验研究,表明COI基因作为我国常见蚊虫分类依据可靠,对复合种团的近似种也能有效区分,但受样本数量限制,对复合种团的分类鉴定还需要进一步收集更多的样品进行确认。口岸蚊类信息化数据平台有蚊虫COI基因鉴定检索功能,从基因水平上为蚊虫鉴定提供了有效手段。基于口岸蚊类信息化数据平台建立的实验室质量控制体系可靠。以标准标本为核心建立的质控体系,通过了CNAS的认可。利用标准化标本,组织了我国首次蚊类形态学鉴定能力验证活动,一方面了解了同类实验室整体技术水平,另一方面也证明了标准化标本作为质控的可靠性。信息化数据平台集成了监测点的地理、生态数据,具有综合分析功能,可自动化、智能化分析,还可以进行预警,为决策提供依据。更多还原

【Abstract】 BackgroundThe threats of mosquito-borne diseases are growing worldwide. For example, West Nile fever, originally restricted in Africa and other tropical regions, has been popular in the United States in1999-2012; Endemic regions of dengue fever and Chikungunya fever have also expanded to many countries. World Health Organization (WHO) estimates that the population infected with dengue virus could reach100million each year. There are outbreaks of dengue fever every year in Guangdong Province, China. In2010, outbreak of Chikungunya fever occurred in Dongguan area of Guangdong Province.No vaccine and specific drug treatment are available for most of mosquito-borne diseases. The main preventive measures for mosquito-borne diseases are to monitor and control mosquitoes. The first step will be the surveillance of mosquitoes, by doing so we can understand the biological vector population composition, density, and seasonal fluctuation. On the other hand, based on the mosquito monitoring data, we can provide forecasting and early warning of the mosquito-borne diseases.The mosquito-borne diseases have their own specific mosquito vectors. Some mosquitoes can not transmit any disease, and some are capable of carrying some virus transmitting to human population. Therefore, the accurate identification of mosquito species is the fundamental of the mosquito surveillance and mosquito-borne disease controlling. Traditional morphological classification relies largely on the subjective judgment and practical experiences of the identifier. Hoever, in most laboratories, experienced morphological identifierer are lacking.Due to the small body size, mosquitos can easily immigrants through containers and ships. During2005-2007The total number of imported mosquitoes added up to288thousands in China. Surveys found that the rate of ship carrying mosquitoes between18%-48%at different ports. But one third of these mosquitoes were not classified at species level. In2008, a total of136episodes of imported infectious diseases were reported in China, including85imported dengue fever cases and5Chikungunya fever cases.Therefore, the port health authorities attach great importance to the mosquito monitoring. On one hand, we can get the accurate knowledge of population composition and seasonal fluctuation of the mosquitoes, and ready to find new kinds of suspicious imported diseases by monitoring mosquitoes in the frontier port area; On the other hand, to carry out mosquito monitoring on the entry and exit of transport, transport equipment, we can establish a health barrier to prevent mosquito-borne diseases spreading through the borders.There are many difficulties in the laboratories of port to carry out the mosquitos’ identification. Due to the lack of professional identification experts, limited reference materials, composite species-group identification is difficult to identify and monitoring and appraisal data can not be shared, also lacking of a quality control system to evaluate the accuracy of mosquito classification and identification in port laboratories. The vector labs of disease control departments in our country also face the same problems.Objective1. To establish the mosquito’s self-identification methods. The retrieve tables and schematic diagrams were widely used reference books for mosquito identification. But the actual specimens under microscope are sometimes different from those included in these books. This study amied to establish a self identification method by using standardized specimens and electronic and digital specimens. 2. To construct the mosquito’s identification methods based on gene identification technology, as an effective complement to the traditional morphological identification method. Gene identification can efficiently identify composite species-group that is hard by traditional morphological methold. And it can be cross-checked with the morphological identification for those species difficult to identify.3. To build up the inter-laboratory information sharing mechanism. The database integrated with data analysis and early warning function based on network technology.4. To set up the inter-laboratory quality control system in the mosquito identification laboratories.Methods1. The establishment of the database of the mosquito specimensThe whole process of a standardized physical adult mosquito specimen includes larvae collecting, laboratory incubators with whole scales, identified by morphological authoritative experts. Then on standardized physical specimens, images of each identification details were acquainted, supplemented by authoritative text description. Stereoscopic camera, combined with the software to the quasi-three-dimensional picture of the electronic synthesizer, digital three-dimensional specimen could be produced.2. The establishment of common mosquitoes genes based on the COI gene databaseWe designed universal primers amplified mitochondrial cytochrome oxidase subunit (COI) for some gene sequences for some collected mosquito species, as well as some complex members that were difficult to identify.DNA sequence was analyzed using EditSeq function module in DNASTAR package. Mosquito homology and phylogenetic analysis to analyze the feasibility of gene identification was done using MegAlign software. On this basis, we established a common mosquito species gene database.3. Data entry and analysis We built up one information platform, where mosquito’s database was a core content which shared the network by interactive design. Each monitoring point or laboratories entered their results. And monitoring data can be shared. Geographic information monitoring points, the basic data of the monitoring point ecological information were added to the system with integrated data analysis capabilities, automatic analysis and graphs, charts. Geographic information, the ecological situation of the density and population of mosquitoes and monitoring point was closely related to the comprehensive ecological monitoring points and the geographic information, this greatly enhanced the efficiency of data analysis.4. The establishment of laboratory quality control systemWe identified as a laboratory internal reference standards by sharing of electronic, digital and genetic database; we built identification of mosquito-borne laboratory quality management system based on ISO:IEC17025.We carried out inter-laboratory quality control by using proficiency testing of standardized physical specimens database. Through information technology databases, the digitized samples could be used as a new method of inter-laboratory quality control based on a network of inter-laboratory comparison.Results1. Eshtablishment of mosquito specimen databaseLarvae samples were collected from the fields and grew up to adult mosquito in the laboratory, serving as the specimens. All the specimens were identified by two authority morphological experts. And the database included standard physical specimens of11genera and59kinds, covering more than2,000common types in frontier ports.A high-resolution digital photograph was taken for each mosquito; the electronic images for adult mosquito specimens were confirmed by authoritative experts. Electronic specimens library content including:identification of mosquito species characterization, digital photographs of each identification details, the details of the electronic text description, ecological habits, and the relationships with infectious diseases. Total components are together to form an electronic mosquito species specimens.5genera and41species of mosquitoes electronic specimens produced, accounting the50%of the species covered by the common mosquito species (population constitutes more than1%) of frontier port has foundWe used the integrated treatment of the web technologies such as JavaScript, DHTML, image synthesis prevail in the three-dimensional pictures to deal with electronic images for all mosquito species,, users can overall observe or just amplifier the picture specified range.2. Established common mosquitoes genes database based on the COI geneWe sequenced adult mosquitoes collected in Pu’er in Yunnan, Guangzhou, Shenzhen, measured a total of14kinds of40adult mosquitoes COI gene sequences. Sequence analysis showed that COI amplified fragments of various mosquito species are the same size, the nucleotides in length415bp. The sequence of the measured strains are no missing, the sequence of the A+T base content range of69.09-71.51%, no missing sequence of each strain. The differences are between97.5-100%homology between the different geographic strains of the same species of mosquito species, and the ranges of homology between the different species are83.5-93.7%. Species whose intraspecific genetic distance is between0-2.5%, the interspecies genetic distance is6.6-18.8%.Mosquito sequence gene cluster analysis found that the first clustering with different geographical strains between then Culex, Aedes, A mosquito is, respectively, clustering, and then the three genera clustering is consistent Culex subfamily, Anopheles clustering different branch, and morphological classification. Composite species-group approximation mosquito species can also effectively distinguish. This shows that the sequence of morphological classification is viable.We set up gene database of36common mosquito species. The database includes three parts of the sequence content, sequence determination method. The users can do sequencing following the way which database provided, or can also check the attached references measures. Determination in the sequence database query window, users can input the COI sequence or part of the sequence of a mosquito, compare with the database. The results could be obtained based on the similarity between the mosquito and the database.3. Information technology database platformIn order to make the database to be better applied, we shared the database to the internal network platform in the Inspection and Quarantine system, so that each laboratory can easily use the database. We designed three types of self-retrieval mode:(1) retrieval table pattern classification retrieval using a binary tree design, structural hierarchy from a species of mosquito retrieve table, each level with a real shot chart and electronic introduction, and by watching the difference between microscope observation and the samples. One can follow a step-by-step procedure with binary tree to do specimen identification.(2) picture comparing retrieval, if the mosquito species is incomplete, or it is difficult to determine according to the steps of the binary tree, based mosquito shape characteristics of a part of the same database matching the selected picture features narrow small range tries to locate the specific species or genera. Users can click to view digitized samples and specific description, can do self identification. Users can also directly call the digitized images to compare.(3) Characteristics retrieval, based on some characteristics of mosquito described its key distinguishing point of the morphological characteristics in the system, in the database retrieval. View of specimens’picture and description by retrieving targeted to certain species, further for self identification.(4) Gene Search, users enter a COI sequence retrieval by comparison, according to the homology from high to low, given the closest three kinds.Information technology database platform compromised integrated monitoring of data entry, monitoring points, geographic information collection, data analysis, quality control. This can be appraisal data sharing through the platform of monitoring. And it can comprehensive analysis of each monitoring point information automatically, generating the corresponding analysis chart, it can also combine with geographic information system, ecological information, monitoring data space warning.4. The application of the database in the inter-laboratory quality controlThe project utilized the established physical specimen, electronic and digitized specimens as a quality control specimen, according to ISO:IE17025system, for the first time, we set up the medical vector laboratory quality control system in Shenzhen Inspection and Quarantine Bureau to take control measures to the equipment which will affect the identification. The system includs four of more than100documents and records form CNAS recognition, and through the recognition.We carried out the morphological identification of adult mosquitos’proficiency testing in China. All the mosquito specimens were double identified by authority experts. A total of28participating laboratories, once through rate was85.7%. The three target mosquito species:Anopheles sinensis, Culex quinquefasciatus, Aedes albopictus identification correct rate of96.2%. Four disturbance mosquito species: harassment A mosquito, Aedes aegypti, the Tritaeniorhynchus Culex, Anopheles minimus identified total accuracy rate is81.2%, including the identification of the correct rate of Anopheles minimus is lowest, at68.5%.ConclusionWe built up a " mosquito’s data platform at port" based on the mosquitoes identification database as a core resource by sharing through the network. The platform compromised with multi media. On the one hand, it can change the current single retrieval table identification way, on the other hand, it can be used as training materials for laboratory identification persons training, as well as the reference and auxiliary tools usually work and improve the identification of the professional level. Using the data platform, identification staffs can be convenient and effective to carry out the work of classification and identification.Verified by experiment, the COI gene can be a reliable basis for identification and classification of common mosquitoes, it can effectively distinguish similar species, but it is limited by the number of samples, whether applicable to other complex species-group classification and identification also need to collect more the number of samples for confirmation.Database based on the laboratory and inter-laboratory quality control system was reliable. The established data quality control standard specimens are under recognition of CNAS. Electronic and digital pictures of database are from the standardized specimens, authoritative, and can be used as the reference laboratory internal quality control routine testing. We organized our first mosquitos’ morphological identification proficiency testing activity by using of the standard specimen, to prove the effectiveness of the quality control system.Information data platform integrates data entry and analysis capabilities. It can be easily shared in the laboratories, and it can analyze comprehensive factors of the monitoring points, geographic information, and ecological information. The system can automatic analyze, and generate charts, tables.更多还原