臺灣博碩士論文加值系統

中文摘要
鳥型分枝桿菌群由緩慢生長的分枝桿菌所組成。很多研究顯示非結核分枝桿菌所引發的臨床表徵以慢性肺部疾病較常見，以最常見的非結核分枝桿菌肺病病原體而言，其順序為M. avium complex、 M. kansasii和M. abscessus。我們發現在2007年7月至2008年12月之間由非結核分枝桿菌所引發的肺部疾病超越了結核分枝桿菌的感染，而其中鳥型分枝桿菌群躍升為非結核分枝桿菌感染中的第一名。期間收集二組痰培養陽性的病人(感染鳥型分枝桿菌群及感染結核分枝桿菌群)各30人，固定性別、診別及居住地，藉此利用「個案對照研究法」分析不同變項 (年齡、身高、體重、BMI、DM、抽菸、肺部病變之位置及是否造成開洞)是否為感染的危險因子。再藉由基因定序比對，找出鳥型分枝桿菌群之Rifampicin的抗藥性基因。 M.avium complex的感染常發生於下肺野(McNemar test﹐p < 0.05)，而開洞(cavity)較常出現在結核分枝桿菌的感染上(Logistic regression﹐p < 0.05)。序列與EMBOSS資料庫M.avium AF060366分析比對發現三株同時在核苷酸序列433位置具點突變(Aspartic acid取代Glycine)。其中體外試驗之表現型對Rifampicin呈抗藥性的有26株(87％)，呈敏感性的有4株(13％)，而基因型對Rifampicin呈抗藥性的約11％，顯現臨床所使用之藥敏試驗應建立一準則，提供能更方便、便宜及精確的體外藥敏試驗。

Abstract
Mycobacterium avium complex (MAC) belongs to a group of slow growing mycobacteria. Many studies showed that chronic pulmonary disease is most commonly manifestated by a non-tuberculous mycobacteria (NTM) infection. MAC, M. kansasii, and M. abscessus, in order of frequency, are the most common pathogens of NTM lung disease. We found the patients with NTM lung disease were more than those with pulmonary tuberculosis between July, 2007 and December, 2008 and MAC was the most common pathogen of NTM lung disease. A case-control was conducted study and 30 patients with a positive sputum culture for MAC as the case group and 30 patients with a positive sputum culture for M. tuberculosis as the control group were enrolled. The two groups were matched with sex, residential areas, and patient services (outpatients or inpatients). Several variables as predictors of risk factors were used to evaluate acquiring NTM lung disease, including age, height, weight﹐BMI﹐DM, smoking, upper lung fields﹐lower lung fields, and pulmonary cavities. We also compared the gene sequence by sequencing to identify the Rifampicin resistance gene of MAC. Involvement of lower lung fields were more common in MAC lung disease (McNemar test, p &lt;0.05), while the pulmonary cavities were more common in pulmonary tuberculosis (Logistic regression, p &lt;0.05). Finally, EMBOSS were used for the database of M.avium AF060366 and sequence analysis. Three strains with a point mutation (Glycine→Aspartic acid) in the nucleotide sequence 433 was noted. Twenty-six strains (87%) presented the phenotype of Rifampicin resistance, while 4 strains were sensitive to Rifampicin (13%). However, genotypes of Rifampicin resistance were noted for only 11% of the strains. It implies that a new criteria for clinical susceptibility test should be established to provide a more convenient, cheap and accurate method for the in-vitro susceptibility test.

目錄
中文摘要............................................................I
英文摘要..........................................................III
本文目錄............................................................V
圖表目錄........................................................ VIII
附錄目錄............................................................X
縮寫表............................................................ XI
第一章 緒論.........................................................1
1-1 研究動機........................................................3
1-2結核分枝桿菌群(Mycobacterium tuberculosis complex)的分類及介紹..................................................................4
1-3結核分枝桿菌群(Mycobacterium tuberculosis complex)其流行病學..................................................................5
1-4非結核分枝桿菌(Nontuberculous mycobacteria)的分類及介紹..................................................................5
1-5非結核分枝桿菌(Nontuberculous mycobacteria)其流行病學................................................................. 6
1-6 非結核性分枝桿菌肺部感染的診斷..................................................................7
1-7 結核與非結核性分枝桿菌的實驗室診斷..............................8
1-8鳥型分枝桿菌群(Mycobacterium avium complex)(MAC)之介紹.................................................................10
1-9 結核病之藥物介紹...............................................11
1-10工作大綱.......................................................12
第二章 材料與方法.................................................14
2-1材料............................................................14
2-1-1試劑......................................................14
2-1-2儀器設備......................................................15
2-2收案對象........................................................16
2-2-1檢體收集......................................................16
2-2-2檢體前處理及培養..............................................17
2-2-3菌株鑑定......................................................18
2-2-4藥物感受性試驗................................................20
2-3分子生物學......................................................21
2-3-1抽取菌株M.avium complex之DNA..................................21
2-3-2 DNA amplification............................................22
2-3-3瓊酯明膠電泳(Agarose gel electrophoresis).....................22
2-3-4核苷酸序列RNA polymerase gene (apo B)定序.....................23
2-3-5核苷酸序列變異分析............................................23
2-4統計分析........................................................23
2-4-1個案對照研究法 (case-control study)...........................23
2-4-2類別資料......................................................24
2-4-3連續資料......................................................25
2-4-4邏輯回歸分析..................................................25
第三章 結果........................................................26
3-1固定項(性別、診別及居住地)在二個族群中之探討....................26
3-2連續變項(年齡、身高、體重、BMI)在二個族群中之探討...............26
3-2-1年齡.........................................................26
3-2-2身高..........................................................26
3-2-3體重..........................................................27
3-2-4 BMI..........................................................27
3-3本研究族群之流行病學表徵........................................27
3-4以邏輯迴歸分析預測罹患M.avium complex之罹病風險.................27
3-5核苷酸定序分析結果..............................................28
第四章 討論........................................................29
第五章 結論........................................................33
參考文獻...........................................................34

附圖與附表
表1-1感染鳥型分枝桿菌群 (MAC) 之收案對象條件探討...................40
表1-2感染結核分枝桿菌群 (TBC) 之收案對象條件探討….................41
表2-1本研究族群之流行病學表徵......................................42
表2-2以邏輯迴歸分析預測罹患M. avium complex之罹病風險..............43
圖1-1結核分枝桿菌與非結核分枝桿菌趨勢圖............................44
圖1-2結核病通報病例人數趨勢圖(96年7月至97年12月)...................45
圖1-3本實驗之主要工作流程..........................................46
圖2-1結核分枝桿菌群 (TBC)，在L-J斜面培養基上為表面粗糙的淡黃色臘狀菌落.................................................................47
圖2-2結核分枝桿菌群 (TBC)(1000X) ..................................48
抗酸性染色其菌體纖細、微彎曲、強抗酸性染色、仔細觀察會帶有明顯的珠狀菌體
圖2-3 Nitrate還原試驗，陽性反應為產生粉紅至紅色顏色變化， 3+至5+為陽性。TBC呈陽性反應.............................................................49
圖2-4 Niacin試驗，陽性反應為產生黃色，TBC呈陽性反應................50
圖2-5鳥型分枝桿菌群(MAC)，在L-J斜面培養基上菌落型態多變可為光滑，透明或不透明，呈圓頂或扁平狀甚至為粗糙狀。本圖型態為光滑，不透明扁平狀...........51
圖2-6鳥型分枝桿菌群 (MAC)(1000X) ..................................52
抗酸性染色其菌體是呈典型短的球桿菌
圖2-7 Tween 80 hydrolysis test，neutral red 於pH 7下呈紅色，為陽性反應。MAC在此項試驗為陰性反應...................................................53
圖2-8 Arylsulfatase test，產生紅色即表示陽性反應。MAC在此項試驗為陰性反應.................................................................54
圖2-9當含藥物Rifampincin (RMP) 1μg/ml的四分格抗生素藥敏培養基和對照組小格皆無菌落生長，既可判讀為敏感性 (Susceptible).............................55
圖2-10而當含藥物Rifampincin (RMP) 1μg/ml的四分格抗生素藥敏培養基和對照組小格皆有菌落生長甚至生長數目在不可數的範圍之內，既可判讀為抗藥性 (Resistant)........................................................56
圖2-11 M.avium complex 之PCR 電泳圖， PCR放大M. avium complex rpoB基因片段，其產物位置為542 bp...................................................57
圖3-1本研究族群固定項(性別、診別及居住地)之探討....................58
圖3-2年齡在盒子圖 (Box Plot) 中的分布狀況..........................59
圖3-3身高在盒子圖 (Box Plot) 中的分布狀況..........................60
圖3-4體重在盒子圖 (Box Plot) 中的分布狀況..........................61
圖3-5 BMI在盒子圖 (Box Plot) 中的分布狀況..........................62
圖3-6罹患MAC與TBC二個族群在X光上之變化.............................63
圖3-7基因定序分析結果…............................................64
附錄
附錄1 TB藥物抑制機轉...............................................65
附錄2 Rifampincin藥物抑制機轉......................................66
附錄3分枝桿菌依生長速度及色素產生情形分群……......................67
附錄4藥物感受性試驗，緩慢生長菌採間接瓊脂比例法 (Indirect proportion method)............................................................68

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