本文選題：隱孢子蟲 + 基因分型�。� 參考：《華東理工大學》2013年博士論文

【摘要】：隱孢子蟲(Crypto sporidium)是一種重要的人獸共患病原體,也是我國和世界多國飲用水水質(zhì)標準中規(guī)定檢測的兩個病原之一。然而,目前隱孢子蟲在水環(huán)境和易感人群中的分布、傳播特征及其溯源研究多在發(fā)達國家開展,在我國少有研究報道。為了解隱孢子蟲在上海地區(qū)不同水環(huán)境中的分布及其來源,本課題采集并濃縮了50份上海黃浦江原水樣品、30份自來水樣品和28份游泳池水樣品,并采用PCR-RFLP(酶切片段長度多態(tài)性分析)方法對樣品中的隱孢子蟲進行了檢測和基因分型。原水中隱孢子蟲的檢出率為28.0%(14/50),包含4個蟲種,分別為牛源的C. andersoni、豬源的C. suis、禽源的C. baileyi和C. meleagridis。其中只有C. meleagridis是感染人的常見蟲種之一,但該蟲種只在一個樣品中發(fā)現(xiàn)。在所有自來水和游泳池水樣品中均未檢測到隱孢子蟲。為快速了解隱孢子蟲在上海地區(qū)的人際傳播特征,本課題采集了上海和三個對照城市南京、青島、武漢的生活污水樣品各90、87、109和100份。通過使用分子檢測和基因分型工具發(fā)現(xiàn),隱孢子蟲在四個城市生活污水中的檢出率分別為70.0%、37.9%、68.8%和47.0%,并且人源蟲種C. hominis在上海污水陽性樣品中檢出率高達93.7%,顯著高于三個對照城市(24.2%、1.3%、40.4%)。另外,上海污水中C. hominis亞型多樣性高,存在I、Ib、Id、Ie、If亞型家族,其中Ib家族中的亞型IbA21G2最為普遍(46.8%),暗示上海城市人群中存在C. hominis密集而穩(wěn)定的傳播,且可能存在由某一種C. hominis亞型導致的隱孢子蟲病暴發(fā)流行。兒童是隱孢子蟲病的易感人群。為明確隱孢子蟲,尤其是C. hominis蟲種,在上海易感兒童中的實際感染和傳播特征,本課題對6284名住院兒童中的隱孢子蟲感染情況進行了研究。在所有兒童中,共有102人(1.6%)被隱孢子蟲感染,感染蟲種分別為C. hominis(92例)、C. meleagridis(6例)、C. canis(2例)和C. felis(2例)。其中從77份C. hominis P日性樣品中發(fā)現(xiàn)6個基因亞型,分別是IdA19(37例)、IaA14R4 (36例)、IbA19G2(1例)、IdA14(1例)、IaA18R4(1例)和IgA14(1例)。由于兩個優(yōu)勢亞型在當?shù)匚鬯胁⑽窗l(fā)現(xiàn),本研究對陽性樣品來源進行了追蹤,結果顯示在采樣醫(yī)院I病區(qū)A的住院兒童中存在隱孢子蟲病的持續(xù)性暴發(fā)。該暴發(fā)由兩個毒力和群體結構各不相同的C. hominis亞型IaA14R4口IdA19主導,其中亞型IaA14R4和腹瀉顯著相關,而IdA19則不然。群體遺傳學分析表明,IaA14R4亞型群體為流行性群體結構,而IdA19亞型群體為克隆性群體結構。本課題進一步對上海住院兒童中隱孢子蟲的感染來源進行了分析和追蹤�；陔[孢子蟲的宿主適應性,本研究發(fā)現(xiàn)散發(fā)病例多涉及人獸共患蟲種,與患兒接觸鳥(禽)類、狗和貓有關。暴發(fā)病例中亞型IaA14R4源自上海某孤兒院,該亞型在課題開展后期依然在孤兒院兒童中廣泛流行。相反,另一個暴發(fā)亞型IdA19很有可能來自醫(yī)院I內(nèi)部。結合其它多個病原的共同傳播特征,本研究推測護理人員在給兒童換尿布和喂食時的不衛(wèi)生操作很可能是導致疾病在兒童中持續(xù)傳播的主要原因。HIV+病人也是隱孢子蟲病的易感人群。本研究發(fā)現(xiàn),隱孢子蟲病在河南HIV+病人中感染率為1.5%(10/683)。感染蟲種除C. hominis(2例)外,其余均為動物源蟲種,如鳥(禽)源的C. meleagridis(5例)、反芻獸源的C. parvum(2例)和豬源的C. suis(1例)。風險因素分析表明,動物-人的傳播為該病原體的主要傳播方式。另外,以井水作為飲用水源是感染該病的風險因素。鑒于基因分型在判斷隱孢子蟲污染和感染源方面的重要性,本研究建立了基于熒光共振能量轉(zhuǎn)移(FRET)雜交探針的兩種LightCycler real-time PCR方法。新建方法可通過熔解曲線分析在同一反應中區(qū)分不同隱孢子蟲蟲種／基因型。其中基于SSU rRNA基因建立的real-time PCR方法可擴增和區(qū)分感染人的5個主要蟲種及水環(huán)境中多數(shù)常見蟲種／基因型,基于HSP90基因的real-time PCR方法能較好地區(qū)分感染人的三個優(yōu)勢蟲種。新建方法在進一步優(yōu)化和標準化后有望成為新一代隱孢子蟲檢測和分型工具。本課題研究結果對于明確隱孢子蟲在我國的公共衛(wèi)生學意義和制定有效的防控策略有重要意義,并對隱孢子蟲基因分型和病原追蹤技術的發(fā)展有積極推動作用。
[Abstract]:Cryptosporidium (Crypto sporidium) is an important zoonotic pathogen and one of the two pathogens in the drinking water quality standards of China and the world. However, the distribution, transmission characteristics and traceability of Cryptosporidium in the water environment and susceptible population are mostly carried out in developed countries. There are few research reports in China. In order to understand the distribution and origin of Cryptosporidium in different water environment in Shanghai, 50 samples of Shanghai Huangpu River, 30 tap water samples and 28 swimming pool water samples were collected and concentrated, and the PCR-RFLP (enzyme cut fragment length polymorphism analysis) method was used to detect and genotyping of Cryptosporidium in the samples. The detection rate of Cryptosporidium in the original water is 28% (14/50), including 4 insect species, C. andersoni of cattle source, C. suis of pig origin, C. baileyi and C. meleagridis. of poultry sources, only C. meleagridis is one of the common species of infected people, but the species is found only in one sample. In all water and swimming pool water samples. Cryptosporidium was not detected. In order to quickly understand the interpersonal transmission characteristics of Cryptosporidium in Shanghai area, we collected 90,87109 and 100 samples of domestic sewage from Shanghai and three control cities, Nanjing, Qingdao and Wuhan. Through the use of molecular detection and genotyping tools, Cryptosporidium was found in four urban sewage. The detection rates were 70%, 37.9%, 68.8% and 47% respectively, and the detection rate of human species C. hominis in Shanghai sewage positive samples was up to 93.7%, significantly higher than that of three control cities (24.2%, 1.3%, 40.4%). In addition, the diversity of C. hominis subtypes in Shanghai sewage was high, and there were I, Ib, Id, Ie, and If subtypes, among which the Ib family subtype IbA21G2 was the most common. (46.8%) (46.8%) suggested that there is a dense and stable transmission of C. hominis in urban population, and there may be a outbreak of Cryptosporidium caused by a C. hominis subtype. Children are susceptible population of Cryptosporidium disease. The actual infection and transmission of Cryptosporidium, especially the C. hominis species, in susceptible children in Shanghai The infection of Cryptosporidium in 6284 hospitalized children was studied. In all children, 102 (1.6%) were infected by Cryptosporidium in all children. The infected species were C. hominis (92 cases), C. meleagridis (6 cases), C. canis (2 cases) and C. felis (2 cases). Among them, 6 subtypes were found from 77 C. hominis P daily samples. IdA19 (37 cases), IaA14R4 (36 cases), IbA19G2 (1 cases), IdA14 (1 cases), IaA18R4 (1 cases) and IgA14 (1 cases). Since two dominant subtypes were not found in local sewage, this study traced the positive sample source. The results showed that there was a persistent outbreak of Cryptosporidium disease in the hospitalized children of A in the hospital I ward. The outbreak was two. The C. hominis subtype IaA14R4 mouth IdA19 dominated by different virulence and population structure, in which subtype IaA14R4 was significantly associated with diarrhea, while IdA19 was not. Population genetic analysis showed that the IaA14R4 subgroup was a popular group structure and IdA19 subgroup was clonogenic. The subject was further hidden in Shanghai inpatient children. The source of the infection of the Sporozoa was analyzed and traced. Based on the host adaptability of Cryptosporidium, this study found that the sporadic cases involved more and more zoonotic species and were associated with birds (birds), dogs and cats. The outbreak cases of Central Asian IaA14R4 originated from an orphanage in Shanghai, and the subtype was still widely used in orphanage children in the later period. In contrast, another outbreak of subtype IdA19 is likely to come from the hospital I. Combined with the common transmission characteristics of many other pathogens, this study speculated that the unhygienic operation of nurses in changing diapers and feeding to children is likely to be the main cause of the disease in children, the main reason for the disease,.HIV+, is Cryptosporidium. This study found that the infection rate of Cryptosporidium in Henan HIV+ patients was 1.5% (10/683). In addition to C. hominis (2 cases), the other species were all animal species, such as C. meleagridis (5 cases) of bird (bird) source, C. parvum (2 cases) from ruminant source and C. suis (1 cases) of pig sources. In addition, well water as a drinking water source is a risk factor for infection of the disease. In view of the importance of genotyping in determining the importance of Cryptosporidium contamination and source of infection, two LightCycler real-time PCR methods based on fluorescence resonance energy transfer (FRET) hybridization probe have been established. The over fusion curve analysis distinguishes different Cryptosporidium species / genotypes in the same reaction. The real-time PCR method based on the SSU rRNA gene can amplify and distinguish 5 main species of the infected people and the most common species / genotypes in the water environment. The real-time PCR method based on the HSP90 gene can distinguish the infected people well. Three dominant species. The new method is expected to be a new generation of Cryptosporidium detection and typing tool after further optimization and standardization. The research results are of great significance for identifying the public health significance of Cryptosporidium in our country and the formulation of effective prevention and control strategies, and the genotyping and pathogen tracking techniques of Cryptosporidium. The development has a positive role in promoting.
【學位授予單位】：華東理工大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R531.5

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相關期刊論文 前1條

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