【摘要】：血吸蟲病是對(duì)人與動(dòng)物危害最嚴(yán)重的寄生蟲病之一,是世界衛(wèi)生組織認(rèn)定的分布范圍最廣、經(jīng)水傳播疾病(Water-borne diseases)感染率最高的寄生蟲病。至2015年底,江蘇省除南京市四個(gè)區(qū)縣(棲霞、六合、浦口、江寧)與鎮(zhèn)江市3個(gè)區(qū)縣(丹徒、揚(yáng)中、潤(rùn)州)尚處于血吸蟲病控制階段,其余各市(縣、區(qū))均已達(dá)血吸蟲病傳播阻斷標(biāo)準(zhǔn)。然而,長(zhǎng)江中上游部分省市仍然處于疫情控制階段,其陽(yáng)性釘螺與感染病人病畜仍時(shí)有發(fā)現(xiàn),部分達(dá)標(biāo)地區(qū)存在釘螺復(fù)燃情況,且釘螺(Oncomelania hupensis)可隨水系擴(kuò)散與人群流動(dòng)頻繁等因素,對(duì)江蘇血防構(gòu)成較大的威脅。因此,對(duì)當(dāng)前低流行態(tài)勢(shì)下的重點(diǎn)區(qū)域血吸蟲病流行情況做一全面調(diào)查,對(duì)血防策略的調(diào)整具有重要意義。血防監(jiān)測(cè)包括對(duì)血吸蟲中間宿主(釘螺)分布、終宿主(人與易感動(dòng)物)的流行病學(xué)調(diào)查、應(yīng)用“哨鼠”開展早期預(yù)警、晚期血吸蟲病人的診斷與治療。在當(dāng)前低流行態(tài)勢(shì)下,以抗體ELISA檢測(cè)、糞便查蟲卵為主的方法,已滿足不了早期監(jiān)測(cè)的要求,亟需發(fā)展快速、準(zhǔn)確的檢測(cè)新方法：寄生蟲學(xué)剖檢查成蟲的哨鼠檢測(cè)方法也存在時(shí)效性差、耗時(shí)耗力、準(zhǔn)確性不高等缺點(diǎn),迫切需要發(fā)展更為準(zhǔn)確、快速、靈敏的方法；再者,針對(duì)新發(fā)展晚血病人與現(xiàn)癥病人的鑒別,在血吸蟲病低流行態(tài)勢(shì)下,需建立新的輔助診斷方法。本研究在南京地區(qū)開展了血吸蟲病流行病學(xué)調(diào)查,并基于血清差異多肽建立診斷新方法以及應(yīng)用于實(shí)驗(yàn)小鼠、預(yù)警“哨鼠”及晚血與現(xiàn)癥病人的鑒別診斷。1.釘螺及其感染尾蚴與人畜感染日本血吸蟲抽樣調(diào)查2013-2014年在棲霞區(qū)、2015年在高淳區(qū)的蕪申運(yùn)河水陽(yáng)江段開展了釘螺密度與人畜感染情況調(diào)查。方法：根據(jù)《血吸蟲病控制與消除標(biāo)準(zhǔn)GB15976-2015》,機(jī)環(huán)結(jié)合法開展釘螺調(diào)查,DDIA血清學(xué)與尼龍絹袋集卵孵化糞檢法開展人群調(diào)查,塑料杯頂管糞便孵化法檢測(cè)動(dòng)物血吸蟲感染；并結(jié)合往年報(bào)表數(shù)據(jù)分析。結(jié)果：棲霞區(qū)與高淳區(qū)的水陽(yáng)江段均未查到陽(yáng)性釘螺：但水陽(yáng)江段活螺密度顯著高于其他地區(qū),提示水陽(yáng)江段具備血吸蟲病傳播風(fēng)險(xiǎn)。人群調(diào)查顯示,棲霞區(qū)2個(gè)行政村的血清學(xué)檢查陽(yáng)性率均低于1%,高淳區(qū)3個(gè)行政村血檢陽(yáng)性率2.48%,糞檢均未查出陽(yáng)性。家畜糞檢未檢測(cè)到感染。結(jié)論：綜合2004-2014江蘇省血吸蟲病防治工作報(bào)表分析,江蘇省內(nèi)有螺面積逐年減少,活螺數(shù)與活螺密度維持在相對(duì)穩(wěn)定的較低水平；血吸蟲病呈低度流行態(tài)勢(shì),近年無(wú)血吸蟲急性感染病例報(bào)道；高淳區(qū)水陽(yáng)江段血檢陽(yáng)性率高于1%的血吸蟲病傳播控制標(biāo)準(zhǔn),也高于全省其他地區(qū)的血清陽(yáng)性率,提示該地區(qū)具有較高風(fēng)險(xiǎn),需加強(qiáng)疫情監(jiān)測(cè)。2.實(shí)驗(yàn)兔血清肽譜分析及基于血血清差異多肽檢測(cè)法的建立分析血吸蟲急性感染實(shí)驗(yàn)兔的血清肽質(zhì)量指紋圖譜,并根據(jù)血清差異多肽建立診斷方法。方法：通過(guò)血清采集、磁珠富集血清多肽、MALDI-TOF/TOF質(zhì)譜檢測(cè)與ClinProTools分析血清肽質(zhì)量指紋圖譜,建立人工感染兔的血清差異多肽診斷方法(CPT方法),并進(jìn)行敏感性與特異性分析,比較多種方法檢測(cè)不同感染時(shí)期的準(zhǔn)確性。結(jié)果：Mr 1787蛋白分子在兔感染后第6d有顯著上調(diào)(P0.05)；在感染后第9 d,Mr 2834蛋白分子有極顯著上調(diào)(P0.01),Mr 3483呈顯著上調(diào)(P0.05),Mr 4018有顯著下調(diào)(P0.05)。在感染后第12 d,Mr 3151蛋白分子有顯著下調(diào)(P0.05),而Mr 4018則出現(xiàn)極顯著下調(diào)(P0.01),Mr 3530呈顯著上調(diào)(P0.05),兩者趨勢(shì)一直持續(xù)至感染后第30 d。經(jīng)二級(jí)質(zhì)譜鑒定并搜庫(kù)比對(duì),預(yù)測(cè)Mr 1787蛋白為α烯醇化酶的片段,Mr 3530預(yù)測(cè)為熱休克蛋白90或熱休克蛋白47片段,提示此兩種蛋白可能為血吸蟲感染兔的早期疾病標(biāo)志物。基于感染第5周與健康對(duì)照組血清差異多肽指紋圖譜,利用ClinProTools軟件建立感染組與健康對(duì)照組的區(qū)別診斷模型,盲測(cè)樣本顯示,感染1、2、3、4 w的家兔血清鑒定為陽(yáng)性的占30%、55%、75%、80%,而同期ELISA檢測(cè)結(jié)果分別為0,0,20%和50%,利用弓形蟲感染血清驗(yàn)證,結(jié)果顯示特異性好。結(jié)論：基于血清差異表達(dá)多肽建立的診斷方法,敏感性要優(yōu)于常規(guī)的ELISA法,有望應(yīng)用于血吸蟲病早期檢測(cè)并篩選具有診斷價(jià)值的血清多肽(蛋白)標(biāo)志物。3.血血清多肽檢測(cè)方法的應(yīng)用3.1血清差異多肽檢測(cè)法在實(shí)驗(yàn)小鼠模型上的應(yīng)用小鼠動(dòng)物模型在血吸蟲感染實(shí)驗(yàn)及相關(guān)研究中應(yīng)用廣泛,發(fā)展對(duì)其快速、準(zhǔn)確、簡(jiǎn)便的診斷方法意義重大。方法：采用血清富集并磁珠純化多肽、MALDI-TOF/TOF質(zhì)譜檢測(cè)與ClinProTools分析肽質(zhì)量指紋圖譜,根據(jù)急性感染第5 w與健康對(duì)照組血清差異表達(dá)多肽,建立檢測(cè)方法(CPT方法),并比較ELISA、寄生蟲學(xué)剖檢等多種方法的檢測(cè)敏感性,并驗(yàn)證急性感染不同時(shí)期、不同感染度小鼠的準(zhǔn)確性。結(jié)果：與傳統(tǒng)的ELISA方法比較,CPT方法在第1w即檢測(cè)到急性感染小鼠5%(3/60)的陽(yáng)性率,在第2w至第5w呈逐漸上升趨勢(shì),分別為35%(21/60),75%(45/60),87.93%(51/58)與98.15%(53/54),而ELISA方法在感染后第3w才檢測(cè)到陽(yáng)性,其3-5 w的感染率分別為65%(39/60),77.59%(45/58)與94.44%(51/54),均低于CPT方法；寄生蟲學(xué)剖檢在第3、4 w有70%(7/10)的小鼠被檢出陽(yáng)性,低于CPT方法同期檢出率。針對(duì)不同感染度小鼠的診斷,在感染14,18,22條尾蚴的小鼠中,所有小鼠均被預(yù)測(cè)為感染,準(zhǔn)確性達(dá)100%,感染4、6、10條尾蚴組的小鼠預(yù)測(cè)準(zhǔn)確率分別為40%(4/10),50%(5/10)與80%(8/10)。結(jié)論：血清差異多肽檢測(cè)法較血清抗體ELISA檢測(cè)法至少提前2-3 w,較寄生蟲學(xué)剖檢極大的降低了工作量,可以用于低度感染狀態(tài)下的實(shí)驗(yàn)小鼠檢測(cè)和樣本初篩,有望為“哨鼠”檢測(cè)提供新的方法。3.2血清差異多肽檢測(cè)法在血防“哨鼠”預(yù)警上的應(yīng)用利用“哨鼠”監(jiān)測(cè)長(zhǎng)江水體血吸蟲感染風(fēng)險(xiǎn),是我省血防預(yù)警工作的重要手段,寄生蟲學(xué)剖檢查成蟲與肝臟蟲卵結(jié)節(jié)是判斷“哨鼠”感染與否的主要手段,但時(shí)效性較差,且耗時(shí)耗力,需要發(fā)展新的快速檢測(cè)方法。方法：剖檢2015年現(xiàn)場(chǎng)試驗(yàn)的660只哨鼠(含棲霞與高淳60只、寧鎮(zhèn)揚(yáng)600只)并采集血清、5例本室保存的經(jīng)解剖查成蟲確診的陽(yáng)性“哨鼠”與50例陰性“哨鼠”血清,經(jīng)血清多肽富集與質(zhì)譜檢測(cè),獲取多肽指紋圖譜,并導(dǎo)入已建立的血清多肽檢測(cè)方法(CPT法)驗(yàn)證。結(jié)果：5例陽(yáng)性“哨鼠”與50例陰性“哨鼠”均可以被準(zhǔn)確診斷,2015年現(xiàn)場(chǎng)試驗(yàn)的660只哨鼠經(jīng)CPT驗(yàn)證為未感染,與寄生蟲學(xué)剖檢結(jié)果一致。結(jié)論：利用血清差異表達(dá)多肽檢測(cè)方法,較常規(guī)“哨鼠”解剖查成蟲方法,節(jié)省人力物力,并可以提早1-2w對(duì)“哨鼠”進(jìn)行初篩,縮短了檢測(cè)血吸蟲感染的“窗口期”可與常規(guī)病原學(xué)檢測(cè)方法聯(lián)合應(yīng)用于“哨鼠”早期預(yù)警。3.3血清差異多肽檢測(cè)法在鑒別晚血病人上的應(yīng)用晚期血吸蟲病人的診斷及治療是血防工作重點(diǎn),尤其在低流行態(tài)勢(shì)下,對(duì)血吸蟲病地方考核更為重要。病原學(xué)檢查與抗體檢測(cè),很難區(qū)別現(xiàn)癥與既往病人。方法：采集晚血、新發(fā)展晚血、晚血治愈、健康對(duì)照四組人群,血清多肽指紋圖譜檢測(cè)并應(yīng)用ClinProTools分析,建立基于新發(fā)晚血組與健康對(duì)照組血清差異多肽的檢測(cè)方法。結(jié)果：晚血與新發(fā)晚血、新發(fā)晚血組中切脾與否在血清肽譜無(wú)顯著差異。與健康對(duì)照組相比,晚血組在Mr 2662,2991,3241,5337與5906處均顯著下調(diào),在Mr 2082與4282處均顯著上調(diào)；而有史治愈組與健康對(duì)照組比較發(fā)現(xiàn),在Mr 2662,4209,5337與5906處顯著下調(diào),2082處顯著上調(diào)。新發(fā)晚血組與有史對(duì)照組相比,在Mr 3241處顯著下調(diào)。提示Mr 3241顯著下調(diào)、4282顯著上調(diào)可以作為判定新發(fā)晚血的指征,二級(jí)質(zhì)譜鑒定并搜庫(kù),推測(cè)該兩種多肽(蛋白)均為免疫球蛋白重鏈可變區(qū)。根據(jù)新發(fā)晚血組與健康對(duì)照組的差異表達(dá)多肽建立的診斷方法,盲測(cè)試驗(yàn)顯示,晚血組、有史治愈組、無(wú)血吸蟲感染組均與晚血病人具有一致的血清肽譜特征,而糞檢陽(yáng)性病人血清有73.3%(11/15)無(wú)法被驗(yàn)證,不能歸于新發(fā)晚血或健康對(duì)照組,水陽(yáng)江段流行病學(xué)調(diào)查獲取的血清抗體陽(yáng)性但糞檢陰性樣本有86.2%(25/29)被驗(yàn)證為晚血組。結(jié)論：血清差異多肽檢測(cè)法可以間接將晚血病例與現(xiàn)癥病例區(qū)分,但后者可能包括既往感染或反復(fù)感染,故有部分仍然被驗(yàn)證為晚血組,提示感染者有進(jìn)一步發(fā)展為晚血病人的可能；水陽(yáng)江段流行病學(xué)調(diào)查人群可能為既往感染,非現(xiàn)癥病人；血清差異多肽檢測(cè)聯(lián)合常規(guī)寄生蟲學(xué)檢查,可以對(duì)血吸蟲流行病學(xué)調(diào)查進(jìn)行初篩。
[Abstract]:Schistosomiasis is one of the most serious parasitic diseases that have the most serious harm to people and animals. It is the most widely distributed WHO identified by the WHO. By the end of 2015, there are four districts and counties in Nanjing (Qixia, Liuhe, Pukou, Jiangning) and 3 districts and counties of Zhenjiang (Dantu, Yangzhong). However, some provinces and cities in the upper and middle reaches of the Yangtze River are still in the stage of epidemic control. However, the positive snails and infected animals are still found in the upper and middle reaches of the Yangtze River, and the reburning of Oncomelania snails in some of the standard areas and the Oncomelania snails (Oncomelania hupensis) can be found. Such factors as the diffusion of water system and the frequent flow of people constitute a great threat to the schistosomiasis in Jiangsu. Therefore, a comprehensive survey of the epidemic situation of schistosomiasis in the key areas under the current low epidemic situation is of great significance to the adjustment of the strategy of schistosomiasis. The epidemiological investigation of animals, the application of the "sentinel" early warning, the diagnosis and treatment of advanced schistosomiasis patients. Under the current low epidemic situation, the method of detecting the eggs with antibody ELISA and fecal egg detection has not met the requirements of early monitoring. It is urgent to develop a fast and accurate new method for detection of adult worms. The detection methods of sentinel mice also have the disadvantages of poor timeliness, time consuming, and low accuracy. It is urgent to develop a more accurate, rapid and sensitive method. Furthermore, a new method of auxiliary diagnosis should be established for the identification of the newly developed late blood patients and the present patients in the low epidemic situation of schistosomiasis. This study has carried out blood in the Nanjing area. The epidemiological investigation of the parasitics and the establishment of a new diagnostic method based on the serum differential polypeptide and the application in the experimental mice. The differential diagnosis of the early warning "sentinel" and the late blood and the present disease patients.1. snail and its infected cercariae and human and animal infection of Schistosoma japonicum in Qixia District in 2013-2014 years, in 2015 in Gaochun District Wushen canal water Yangjiang A survey on the density of Oncomelania snails and human and animal infection was carried out in the section. Methods: according to the control and elimination standard of schistosomiasis and elimination standard GB15976-2015>, the investigation of Oncomelania snails, DDIA serology and nylon spun hatching fecal test were carried out to conduct a population survey, and the hatching method of plastic cup pipe jacking excrement was used to detect the infection of Schistosoma japonicum. Data analysis. Results: there were no positive Oncomelania snails in the water Yangjiang section of Qixia and Gaochun areas, but the density of living snail was significantly higher in the Shui Yang section than in other areas, suggesting the risk of schistosomiasis transmission in the Shui Yang section. The survey showed that the positive rate of the serological examination of the 2 administrative villages in Qixia was lower than 1%, and the blood test of 3 administrative villages in Gaochun area was positive. The sex rate was 2.48%, the fecal examination was not detected. Conclusion: comprehensive 2004-2014 Jiangsu province schistosomiasis prevention and control work report analysis, the snail area in Jiangsu province is decreasing year by year, the number of living snails and living snail density maintain relatively low level relatively stable; schistosomiasis is low epidemic situation, and in recent years there is no schistosomiasis sensual sex. The infection cases reported that the positive rate of blood test in the Gaochun district was higher than 1% of the standard of schistosomiasis transmission control. It was also higher than the positive rate in other regions of the province, suggesting that the region has high risk. It is necessary to strengthen the analysis of the serum peptide spectrum of the.2. experimental rabbit and the analysis of the blood serum differential polypeptide detection method based on the detection of blood serum differential polypeptide. The quality fingerprint of the serum peptide of the experimental rabbits was dyed and the diagnostic method was established according to the serum polypeptide. Methods: the serum peptide was enriched by the serum collection, the magnetic beads were enriched and the quality fingerprint of the serum peptide was detected by MALDI-TOF/TOF mass spectrometry and ClinProTools analysis, and the diagnostic method of serum differential polypeptide (CPT method) was established. A variety of methods were used to detect the accuracy of different infection periods. Results: Mr 1787 protein molecules were significantly up-regulated (P0.05) in 6D after infection, and at ninth d after infection, Mr 2834 protein molecules were significantly up-regulated (P0.01), Mr 3483 was significantly up (P0.05), Mr 4018 was significantly down (P0.05). Twelfth d after infection, Mr 31 The 51 protein molecules had a significant downregulation (P0.05), while Mr 4018 had a very significant downregulation (P0.01), and Mr 3530 was significantly up-regulated (P0.05). The two trends continued until the infection thirtieth D. were identified by two mass spectrometry and searched the ratio. The Mr 1787 protein was predicted to be an alpha enolase fragment, Mr 3530 was predicted as a heat shock protein 90 or a heat shock protein 47 fragment. The two proteins may be an early disease marker of rabbits infected by Schistosoma japonicum. Based on the differential peptide fingerprint of the serum of the healthy control group for fifth weeks and the healthy control group, the differential diagnosis model of the infection group and the healthy control group was established by ClinProTools software. The blind samples showed that the sera of the rabbits infected with 1,2,3,4 w accounted for 30%, 55%, 75%, respectively. 80%, and the results of ELISA detection in the same period were 0,0,20% and 50% respectively. The results showed that the specificity of Toxoplasma infection was good. Conclusion: the diagnostic method based on the establishment of serum differential expression polypeptide is better than the conventional ELISA method. It is expected to be applied to the early detection of schistosomiasis and screening the diagnostic value of serum polypeptide (protein). Application of marker.3. blood serum polypeptide detection method 3.1 The Application of serum differential polypeptide detection method in experimental mice model is widely used in the experiment of Schistosoma infection and related research. It is of great significance to develop a rapid, accurate and simple diagnostic method for its rapid, accurate and simple diagnostic methods. TOF/TOF mass spectrometry detection and ClinProTools analysis peptide mass fingerprint, according to the acute infection fifth W and the healthy control group serum differential expression polypeptide, establish the detection method (CPT method), and compare the ELISA, the Parasitology examination and so on various methods detection sensitivity, and verify the acute infection different period, the different infection degree mice accuracy. Result: compared with the traditional ELISA method, the CPT method detected the positive rate of 5% (3/60) in acute infection mice at 1W, and gradually increased in 2W to 5W, 35% (21/60), 75% (45/60), 87.93% (51/58) and 98.15% (53/54), while ELISA method was positive in the post infected 3W. The 3-5 infection rate was 65% (77.59%), 77.59% respectively. (45/58) and 94.44% (51/54) were lower than the CPT method; parasitology was detected in 70% (7/10) mice in 3,4 W, lower than the CPT method in the same period. In mice infected with different infection degrees, all mice infected with cercariae were predicted to be infected with the accuracy of 100%, infected with the small cercariae group. The accuracy rate of rat prediction was 40% (4/10), 50% (5/10) and 80% (8/10). Conclusion: the serum differential polypeptide detection method is at least 2-3 w ahead of the serum antibody ELISA detection method, which can greatly reduce the workload and can be used for the detection of experimental rats and the initial screening of samples under the condition of low degree infection. It is expected to provide a new method for the detection of "sentinel". Methods the application of.3.2 serum differential polypeptide detection method in the early warning of schistosomiasis "sentinel" was used to monitor the risk of Schistosoma infection in the Yangtze River. It is an important means for the early warning of schistosomiasis in the Yangtze River. The Parasitology examination of adult and liver egg nodules is the main means to judge whether the "sentinel" is infected or not, but the timeliness is poor, and A new rapid detection method is needed to develop a new rapid detection method. Methods: 660 sentinel mice (including 60 Qixia and Gaochun, Ningzhenyang, Ningzhenyang) in 2015 were examined and serum was collected. 5 cases of positive "sentinel" and 50 negative "sentinel" serums, which were confirmed by anatomic adults, were preserved in this room, and were detected by serum polypeptide enrichment and mass spectrometry. The peptide fingerprint was used and the established serum polypeptide detection method (CPT method) was introduced. Results: 5 cases of positive "sentinel" and 50 negative "sentinel" could be accurately diagnosed. In 2015, 660 sentinel mice were tested by CPT as uninfected and consistent with the results of parasitic examination. The method, compared with the conventional "sentinel" dissection method, saves manpower and material resources, and can early screen the "sentinel" by 1-2W, shortening the "window period" of detecting schistosomiasis infection, which can be applied to the early warning of "sentinel" in the early warning of "sentinel".3.3 serum differential polypeptide detection method in the identification of late blood patients. The diagnosis and treatment of patients with advanced Schistosoma is the focus of schistosomiasis, especially in the low epidemic situation, it is more important for the local examination of schistosomiasis. It is difficult to distinguish between the present and the past patients by the pathogenic examination and the antibody test. Methods: collecting late blood, new development of late blood, late blood cure, healthy control four groups, serum polypeptide fingerprint atlas inspection ClinProTools analysis was used to establish a method for the detection of serum differential peptide in the new late blood group and the healthy control group. The results showed that there was no significant difference in the serum peptide spectrum between the late blood and the new late blood and the new late blood group. Compared with the healthy control group, the late blood group decreased significantly at Mr 2662299132415337 and 5906, at Mr 2082. Compared with the healthy control group, there was a significant decrease in Mr 266242095337 and 5906, and the 2082 up regulated significantly in Mr 266242095337 and 5906. The new late blood group was significantly lower in the Mr 3241 than in the history control group, suggesting that the Mr 3241 was significantly down, and the 4282 up up could be used as a indication of the new late blood and two grade. The two peptides (proteins) were all the variable regions of the immunoglobulin heavy chain. According to the diagnostic method of the differential expression of polypeptide in the new late blood group and the healthy control group, the blind test showed that the late blood group had a history cure group and the non schistosomiasis infection group had the same serum peptide spectrum characteristics with the late blood patients, and the fecal test was positive. 73.3% (11/15) of the sera of the sex patients could not be verified, and could not be attributed to the new late blood or the healthy control group. The serum antibody positive of the water Yang river section was positive but 86.2% (25/29) of the negative samples of the feces was confirmed as the late blood group. Conclusion: the serum differential polypeptide detection method can be used to distinguish the late blood cases from the present cases indirectly, but the latter is the latter. It may include previous infection or recurrent infection, and some of them are still proved to be late blood groups, suggesting the possibility of further development of late blood patients; the epidemiologic survey of the Shui Yang section may be a previous infection, not a present, and a serum differential polypeptide detection combined with a regular parasitic examination can be used for the epidemic of schistosomiasis. A preliminary screening of the study is carried out.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R532.21

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相關(guān)期刊論文 前1條

1 仇志琴;黃玉政;陶永輝;張英;虞豐;;血清差異表達(dá)多肽譜用于人卵巢癌診斷的研究[J];南京醫(yī)科大學(xué)學(xué)報(bào)(自然科學(xué)版);2012年07期

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本文編號(hào)：2128620