隐孢子虫在我国部分地区水环境和易感人群中的分布、传播及其溯源

发布时间：2018-06-21 06:00

本文选题：隐孢子虫 + 基因分型　；参考：《华东理工大学》2013年博士论文

【摘要】：隐孢子虫(Crypto sporidium)是一种重要的人兽共患病原体,也是我国和世界多国饮用水水质标准中规定检测的两个病原之一。然而,目前隐孢子虫在水环境和易感人群中的分布、传播特征及其溯源研究多在发达国家开展,在我国少有研究报道。为了解隐孢子虫在上海地区不同水环境中的分布及其来源,本课题采集并浓缩了50份上海黄浦江原水样品、30份自来水样品和28份游泳池水样品,并采用PCR-RFLP(酶切片段长度多态性分析)方法对样品中的隐孢子虫进行了检测和基因分型。原水中隐孢子虫的检出率为28.0%(14/50),包含4个虫种,分别为牛源的C. andersoni、猪源的C. suis、禽源的C. baileyi和C. meleagridis。其中只有C. meleagridis是感染人的常见虫种之一,但该虫种只在一个样品中发现。在所有自来水和游泳池水样品中均未检测到隐孢子虫。为快速了解隐孢子虫在上海地区的人际传播特征,本课题采集了上海和三个对照城市南京、青岛、武汉的生活污水样品各90、87、109和100份。通过使用分子检测和基因分型工具发现,隐孢子虫在四个城市生活污水中的检出率分别为70.0%、37.9%、68.8%和47.0%,并且人源虫种C. hominis在上海污水阳性样品中检出率高达93.7%,显著高于三个对照城市(24.2%、1.3%、40.4%)。另外,上海污水中C. hominis亚型多样性高,存在I、Ib、Id、Ie、If亚型家族,其中Ib家族中的亚型IbA21G2最为普遍(46.8%),暗示上海城市人群中存在C. hominis密集而稳定的传播,且可能存在由某一种C. hominis亚型导致的隐孢子虫病暴发流行。儿童是隐孢子虫病的易感人群。为明确隐孢子虫,尤其是C. hominis虫种,在上海易感儿童中的实际感染和传播特征,本课题对6284名住院儿童中的隐孢子虫感染情况进行了研究。在所有儿童中,共有102人(1.6%)被隐孢子虫感染,感染虫种分别为C. hominis(92例)、C. meleagridis(6例)、C. canis(2例)和C. felis(2例)。其中从77份C. hominis P日性样品中发现6个基因亚型,分别是IdA19(37例)、IaA14R4 (36例)、IbA19G2(1例)、IdA14(1例)、IaA18R4(1例)和IgA14(1例)。由于两个优势亚型在当地污水中并未发现,本研究对阳性样品来源进行了追踪,结果显示在采样医院I病区A的住院儿童中存在隐孢子虫病的持续性暴发。该暴发由两个毒力和群体结构各不相同的C. hominis亚型IaA14R4口IdA19主导,其中亚型IaA14R4和腹泻显著相关,而IdA19则不然。群体遗传学分析表明,IaA14R4亚型群体为流行性群体结构,而IdA19亚型群体为克隆性群体结构。本课题进一步对上海住院儿童中隐孢子虫的感染来源进行了分析和追踪。基于隐孢子虫的宿主适应性,本研究发现散发病例多涉及人兽共患虫种,与患儿接触鸟(禽)类、狗和猫有关。暴发病例中亚型IaA14R4源自上海某孤儿院,该亚型在课题开展后期依然在孤儿院儿童中广泛流行。相反,另一个暴发亚型IdA19很有可能来自医院I内部。结合其它多个病原的共同传播特征,本研究推测护理人员在给儿童换尿布和喂食时的不卫生操作很可能是导致疾病在儿童中持续传播的主要原因。HIV+病人也是隐孢子虫病的易感人群。本研究发现,隐孢子虫病在河南HIV+病人中感染率为1.5%(10/683)。感染虫种除C. hominis(2例)外,其余均为动物源虫种,如鸟(禽)源的C. meleagridis(5例)、反刍兽源的C. parvum(2例)和猪源的C. suis(1例)。风险因素分析表明,动物-人的传播为该病原体的主要传播方式。另外,以井水作为饮用水源是感染该病的风险因素。鉴于基因分型在判断隐孢子虫污染和感染源方面的重要性,本研究建立了基于荧光共振能量转移(FRET)杂交探针的两种LightCycler real-time PCR方法。新建方法可通过熔解曲线分析在同一反应中区分不同隐孢子虫虫种／基因型。其中基于SSU rRNA基因建立的real-time PCR方法可扩增和区分感染人的5个主要虫种及水环境中多数常见虫种／基因型,基于HSP90基因的real-time PCR方法能较好地区分感染人的三个优势虫种。新建方法在进一步优化和标准化后有望成为新一代隐孢子虫检测和分型工具。本课题研究结果对于明确隐孢子虫在我国的公共卫生学意义和制定有效的防控策略有重要意义,并对隐孢子虫基因分型和病原追踪技术的发展有积极推动作用。
[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.
【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R531.5

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