腸病毒

腸病毒 71 型血清流行病學資料庫之建立及中和試驗替代方法之探討

Studies for establishing a seroepidemiological database of

Enterovirus 71 and evaluating the feasibility of the replacement of

neutralization test

中文摘要 本研究首先針對台灣本島北部之台北市、中部之彰化縣及南投縣、東部之花蓮 縣、南部之高雄市以及離島之澎湖縣等六縣市之 0 ~ 6 個月新生兒、7 個月~ 6 歲 學齡前幼童、7 ~ 15 歲學齡兒童、懷孕婦女及適婚年齡婦女等五個族群共 981 例 血清檢體，利用中和試驗( Neutralization Test；NT )進行腸病毒 71 型抗體力價檢 測。藉由分析抗體陽轉率後發現，新生兒體內來自於母體( maternal )之抗腸病毒 71 型抗體在 6 個月之後會逐漸消失，而 7 個月~ 6 歲學齡前幼童之抗體陽轉率 ( 21.5 % )較 7 ~ 15 歲學齡兒童( 55.6 % )為低，顯示了六歲以下族群為腸病毒感染 之高危險群故應為腸病毒防治之主要對象；另經由統計結果顯示，男性與女性受 腸病毒 71 型感染之比例並無統計上之顯著差異；各項統計結果不論是就個別縣 市或是以都市化程度及城鄉差距劃分後進行分析皆出現相似之情形。此外，在本 研究中亦探討了利用酵素連結免疫吸附測定法( Enzyme - linked Immunosorbent Assay；ELISA )取代中和試驗之可行性，藉由 ELISA 檢測各族群中所含之抗腸 病毒 71 型抗體，並與利用中和試驗所分析出之數據比較；由實驗結果顯示，因 血清中含有多種其他不同類型腸病毒群之抗體，而這些非抗腸病毒 71 型之抗體 會與腸病毒 71 型發生交叉反應 ( Cross — reaction )，因而導致無法利用 ELISA 明確的區別出抗體陽性及抗體陰性之血清檢體；而另外以克沙奇病毒 A16 型進 行與腸病毒 71 型相同之 ELISA 實驗，更加確定了血清中具明顯之抗體交叉反應 干擾。為了進一步證實存在血清中不同類型之腸病毒抗體確實會與不同血清型之 腸病毒群發生交叉反應，因此利用免疫螢光染色分析法( Immunofluorescence assay；IFA )分別針對十三種不同型別之腸病毒進行分析，由實驗結果證實，血 清中含有之各種不同類型腸病毒抗體確實會與腸病毒 71 型及腸病毒 71 型以外之 其他不同型別腸病毒發生交叉反應，因而導致無法利用 ELISA 定量血清中所含 之抗腸病毒 71 型抗體甚至無法進行定性分析，因此在尚未有效的改善腸病毒抗 體間所存在之交叉反應干擾前，目前 ELISA 並不適用於取代傳統檢測腸病毒抗 體力價之中和試驗。 英文摘要

A total of 981 human serum samples consist the children who were less than 6 months old, 7 months through 6 years old, 7 through 15 years old, and the adult females who were fertile and who were pregnant at the time of sampling. These samples, were collected from 6 different areas of Taiwan ( the city of Taipei, the city of Kao-Hsiung,

Chang-Hua County, Nan-Tou County, Hua-lien County, and Peng-Hu County ), and were analyzed for the titer of Enterovirus 71 antibody by using Neutralization Test (NT). The results showed that the newborns’ Enterovirus 71 antibodies coming from maternal would fade away after 6 months. The percentage of positive samples of Enterovirus 71 antibody among the 7 months through 6 years old (21.5 %) was significant lower than that among the 7 years through 15 years old (55.6 %). This results indicate the children under 6 years old have a higher risk in Enterovirus 71 infection and should be the target population. Concerning the gender difference in the infection of Enterovirus 71, no significant difference was found. In addition, no significant difference in terms of the percentage of positive samples of Enterovirus 71 antibody and the degree of urbanization, was found across the aforementioned areas. On the other hand, the feasibility of replacing the NT with the Enzyme-linked Immunosorbent Assay (ELISA) was evaluated by comparing the titer readings

obtained from using ELISA against those of using NT. We found that ELISA failed to discriminate the positive samples from the negative ones, because of the

cross-reaction taking place between the Enterovirus 71 and other types of Enterovirus antibody in a serum sample. A similar result was discovered when the same procedure was conducted on Coxsackievirus A16. The existence of cross-reaction was further confirmed when another 13 types of Enterovirus were analyzed by conducting the Immunofluorescence Assay (IFA). Based on these analyses, they seems indicated that the ELISA is not suitable a substitution for the NT in detecting the titer of Enterovirus antibody unless an effective measurement to avoid the cross-reaction.