人芽囊原蟲是一種流行於全世界的腸道原蟲類寄生蟲，對於人類感染的致病性並不一致，可能與不同的亞型有關，根據其小次單元核醣體基因可被分為17種亞型。分析南部某區域醫院糞便寄生蟲檢驗陽性結果發現，其中人芽囊原蟲檢出率比例高達90.2%，並且外籍人士的感染率明顯高於本國人士（14.7% vs 4.2%）。本研究主要目的想建立巢式分子聚合酶連鎖反應檢測人芽囊原蟲的小次單元核醣體基因並進行基因分型，結果發現在顯微鏡鏡檢陽性的112件檢體中，巢式分子聚合酶連鎖反應有81件為陽性；但在鏡檢陰性的200件檢體中，巢式分子聚合酶連鎖反應卻證實18件為陽性，因此利用巢式分子聚合酶連鎖反應檢測的靈敏性為72.3%，特異性為91%。因此如果利用分生法偵測人芽囊原蟲將可提高9%的偵測陽性率，因此建議臨床實驗室將來可以利用巢式分子聚合酶連鎖反應篩檢人芽囊原蟲的感染。接著將99件分子聚合酶連鎖反應人芽囊原蟲陽性的產物進行定序分型，發現其中最多的分型為ST3（44.4%，44/99）以及ST1（43.4%，43/99），其次還有ST2（5.1%，5/99）、ST6（4.0%，4/99）和ST7（3.0%，3/99）。進一步利用Kimura-2-Parameter模型，採用neighbor-joining方式排序，發現在五種各自的亞型(ST1，ST2，ST3，ST6和ST7)都可以被視為五個獨立的單系群，並在演化樹分析發現Bh97和ST1(AB107961，宿主：豬)及Bh149和ST3(AB107965，宿主：牛)有高度相關，結果可以推斷人芽囊原蟲的感染途徑有人傳人及人畜共通傳染的可能性。另外研究結果發現，目前台灣本國人的人芽囊原蟲感染率雖然低於外籍人士，但台灣人所測到的人芽囊原蟲亞型分布與印尼的流行分布非常相似，可以證實當外籍工作者進入到台灣的家庭中，可能藉由生活接觸造成人芽囊原蟲的傳染。因此對外籍勞工進行適當的衛生教育與治療，才能降低人芽囊原蟲傳染和散播的可能性。

Blastocystis hominis (B. hominis) is one of the most common intestinal protozoan parasites in humans. Pathogenicity of B. hominis remains controversial and it has been suggested that it may be associated with certain subtypes of organism. Based on the genetic diversity of SSU rRNA (small subunit ribosomal RNA gene, SSU rRNA), B. hominis can be classified into 17 subtypes. From the results of stool samples for parasite test from the regional hospital in southern Taiwan, it was demonstrated that B hominis had the highest prevalence rate (90.2%) of intestinal parasites. The infection rate of B. hominis in foreign population was significantly higher than that of nationals (14.7% vs 4.2%). The aims of this study were to set up nested polymerase chain reaction (nested PCR) for DNA amplification and subtyping. The results were shown 81 of 112 specimens positive for B. hominis and 18 of 200 specimens negative for microscopy were shown positive by nested PCR detection. The sensitivity and the specificity of the nested PCR molecular detection method were 72.3% and 91%; respectively. Our results revealed the positive detection rate increased 9% by nested PCR compared with by microscopy. We hoped that this technology can be applied in the clinical laboratory for B. hominis screening. The sequences of 99 PCR positive samples were analyzed. The most common subtypes were ST3 (44.4%, 44/99), ST1 (43.4%, 43/99), followed by ST2 (5.1 %, 5/99), ST6 (4.0%, 4/99) and ST7 (3.0%, 3/99). A phylogenetic tree was reconstructed using the neighbor-joining method based on the Kimura-2-Parameter model and it was found that five subtypes (ST1, ST2, ST3, ST6 and ST7) can be regarded as five independent monophyletic groups. By phylogenetic analysis, Bh97 and ST1 (AB107961, host: pig), Bh149 and ST3 (AB107965, host: cattle) were highly correlated. It can be inferred that human Blastocysts is transmitted by humans and the possibility of common infections between humans and animals. Although the current prevalence of B. hominis in Taiwanese is lower than that of foreign workers, there is still the possibility of mutual infection through fecal-oral route. In this study, we found the subtype distribution of B. hominis were very similar between Taiwan population and Indonesians. Therefore, the foreign workers should be conducted appropriate health education to reduce B. hominis transmission and dissemination.

目錄
論文審定書………………………...………………………………….........i
中文摘要…………………………...…………………………………........ii
英文摘要……………………………………………………..……....……iii
前言…………………………………………………………………......….1
目的…………………………………………………………………….....11
材料與方法………………………………………………….....................12
結果………………………………………………………….....................17
討論………………………………………………………….....................20
參考文獻………………………….………………………………...…….25
圖表……………………………………………………….……...……….38
附錄………………………………………………………….....................57
圖次
圖 1. 本研究檢體收集流程圖………………………………..…...…….38
圖 2. 收集糞便檢體之個案年齡分布…………………………….…….39
圖 3. 收集糞便檢體之個案國籍分布…………………………..…...….40
圖 4. 巢式分子聚合酶連鎖反應檢測結果………..…………….……...41
圖 5. 巢式分子聚合酶連鎖反應檢測結果………..…………….……...42
圖 6. 人芽囊原蟲陽性檢體的基因型分布…………………………......43
圖 7. 人芽囊原蟲的基因序列演化樹分析………………..……...…….44
圖 8. 人芽囊原蟲的ST1亞型基因序列演化樹…………………...…...45
圖 9. 人芽囊原蟲的ST2亞型基因序列演化樹…………………….….46
圖 10. 人芽囊原蟲的ST3亞型基因序列演化樹……………………....47
圖 11. 人芽囊原蟲的ST6亞型基因序列演化樹………………………48
圖 12. 人芽囊原蟲的ST7亞型基因序列演化樹………………………49
表次
表 1. 巢式分子聚合酶連鎖反應使用的特異性引子……......................50
表 2. 糞便濃縮法檢驗腸道寄生蟲感染陽性結……………………......51
表 3. 比較本國籍與外國籍檢體寄生蟲感染的分布………………......52
表 4. 收集312件糞便檢體之個案性別分布………………………..….53
表 5. 巢式分子聚合酶連鎖反應與顯微鏡鏡檢法偵測人芽囊原蟲的比較…….........................................................................................................54
表 6. 不同國籍感染人芽囊原蟲之基因分型………………….…….…55
表 7. 基因庫中Blastocystis spp.的參考序列……………………...…....56
附錄
附圖1. 人芽囊原蟲的生活史與傳染型態…………………………..….57
附圖2. 人芽囊原蟲的致病機轉……………………………..………….58
附圖3. 經由MIF染色的人芽囊原蟲………………………………..….59

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