The Establishment Of Mosquitoes Network Database And The Application In Laboratory Quality Control At Port

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.