臺灣博碩士論文加值系統

對於罹患癌症、接受器官移植和外科手術及燙傷病人，長期接受抗生素治療或AIDS的病患來說，免疫力的下降易伴隨酵母菌的感染。真菌菌血症(fungemia)之死亡率高達35%，是一般細菌菌血症(bacteremia)的二倍以上。抗真菌藥物之最低抑菌濃度(minimum inhibitory concentration，MIC)試驗, National Committee for Clinical Laboratory Standards (NCCLS)建議使用試管稀釋法需 48 小時。本實驗欲利用電容量測法(capacitance measurement method)測定抗真菌藥物之MIC。四種抗真菌藥物(amphotericin B, flucytosine, fluconazole, ketoconazole)對 10株酵母菌在不同時間作5次的MIC測定以探討其再現性。在3 log2 範圍內再現性為98% (amphotericin B, fluconazole, ketoconazole)到100% (flucytosine)；與傳統方法在 2 log2範圍內的吻合度(agreement)為90%。以51個陽性血瓶以直接接種方式對上述四種抗真菌藥物進行MIC測定。此外，在菌株分離後以傳統方法測試MIC，結果顯示各藥物的吻合度( 2log2)分別為amphotericin B (98%), flucytosine (82%), fluconazole (96%), ketoconazole (92%)。若以電容量測法直接進行陽性血瓶之藥物感受性試驗，並以區分點(breakpoint)為測試濃度，據以判斷菌株為susceptible, intermediate (or susceptible-dose dependent )或resistant ，其結果並和傳統的MIC比較。在測試75個陽性血瓶後，電容法的major error為1.3%，minor error為3%。電容量測法對於抗真菌藥物之敏感性試驗為一種新方法，可以免除測定吸光值的繁瑣步驟，也可直接測定陽性血瓶中酵母菌的藥物感受性其結果與傳統方法相關性良好，而可以節省一至二天的分析時間

Severely immunocompromised patients due to AIDS, cancer chemotherapy, organ transplantation, surgery, and burn are predisposed to yeast infections. The mortality rate associated with fungemia is round 35%, this is about twice of that caused by bacteremia. Minimum inhibitory concentrations of antifungal agents are conventional determined by the macrodilution as recommended by the National Committee for Clinical laboratories Standards (NCCLS). In this study, we tried to used a capacitance method for the determination of the MICs of antifungal agents. To test the reproducibility of the capacitance method, the MICs of four antifungal agents (amphotericin B, flucytosine, fluconazole, and ketoconazole) against 10 yeast strains were determined by the method, with each strain being tested five times against each drug on different days. Overall, 3 log2-dilution ranges encompassed 98% of the MIC values of amphotericin B, fluconazole, and ketoconazole and 100% of flucytosine.
Furthermore, we tried to perform direct antifungal susceptibility test of positive blood cultures containing yeasts, The MICs of the above four antifungal agents against 51 blood isolates were determined by the capacitance method and the conventional macrodilution method, the agreement ( 2log2) between the two method were flucytosine (82%), ketoconazole (92%), fluconazole (96%), and amphotericin B (98%). By the capacitance method, direct antifungal susceptibility testing was also performed in a format of the two breakpoint concentrations of flucytosine and fluconazole. The MICs of the two antifungals against the blood isolates were also determined by the NCCLS method. After testing 75 positive blood cultures, capacitance method had a minor error of 3%, and a major error of 1.3%.
The capacitance method is a new method for antifungal susceptibility testing and may be useful for rapid detection of antifungal resistance in positive blood cultures containing yeasts.

1.1真菌的定義與特性-----------------------------------01
1.2真菌感染的一般狀況---------------------------------02
1.3酵母菌及類酵母菌-----------------------------------03
1.3.1伺機性感染真菌簡介-------------------------------03
1.4院內感染情形---------------------------------------06
1.5真菌菌血症感染趨勢---------------------------------08
1.6藥物敏感性試驗法-----------------------------------11
1.6.1傳統檢驗法---------------------------------------11
1.6.2呈色法-------------------------------------------12
1.6.3商品化的Etest------------------------------------12
1.6.4電學法-------------------------------------------13
1.6.5流體細胞計數法-----------------------------------13
1.7本研究之架構---------------------------------------14
1.8電化學法檢測的發展與原理---------------------------16
1.9電容量測法原理簡介---------------------------------19
1.9.1電極電雙層---------------------------------------19
1.9.2電雙層的電容量-----------------------------------24
1.9.3 本實驗中相關的化學物質及藥物作用機制簡介--------29
1.9.4實驗設備簡介-------------------------------------31
第二章 實驗
2.1抗真菌藥物-----------------------------------------32
2.1.1來源---------------------------------------------32
2.1.2抗真菌藥物的秤重---------------------------------32
2.1.3高濃度藥液的配置---------------------------------32
2.2 MIC測試步驟---------------------------------------35
2.2.1合成培養液---------------------------------------35
2.2.2緩衝液-------------------------------------------35
2.2.3材料需求-----------------------------------------35
2.2.4接種源(inoculum)的製備---------------------------36
2.2.5 MIC的測定---------------------------------------36
2.2.6各種藥品廠牌的規格-------------------------------37
2.3 Etest實驗-----------------------------------------38
2.3.1 Etest固體培養基之配方---------------------------38
2.3.2固體培養基配置步驟-------------------------------38
2.3.3接種源準備---------------------------------------39
2.3.4實驗材料-----------------------------------------39
2.3.5實驗步驟-----------------------------------------39
2.4以血液直接進行Etest實驗----------------------------40
2.4.1實驗步驟-----------------------------------------40
2.5以電容量量測法量測MIC------------------------------41
2.5.1藥物製備方式-------------------------------------41
2.5.2材料需求-----------------------------------------41
2.5.3測試步驟-----------------------------------------41
2.6以血液直接進行MIC電容量法量測法--------------------42
2.6.1藥物製備方式-------------------------------------42
2.6.2材料需求-----------------------------------------42
2.6.3測試步驟-----------------------------------------42
2.7檔案傳輸-------------------------------------------43
2.7.1實驗需求-----------------------------------------43
2.7.2實驗步驟-----------------------------------------43
第三章 結果與討論-------------------------------------47
3.1酵母菌生長代謝之量測-------------------------------47
3.1.1酵母菌生長代謝之量測訊號的選取-------------------47
3.1.2 RPMI 1640加入2% 葡萄醣對量測訊號的影響----------50
3.1.3電容量量測法中MIC之判定--------------------------52
3.2電容量量測法的再現性-------------------------------54
3.2.1 以10株酵母測定amphotericin B之MIC（5次實驗)----- 54
3.2.2 以10株酵母測定ketoconazole之MIC（5次實驗)------- 56
3.2.3 以10株酵母測定flucytosine之MIC（5次實驗）-------57
3.2.4 以10株酵母測定fluconazole之MIC（5次實驗）--------58
3.2.5電容量測法與傳統試管稀釋法的比較-----------------59
3.3菌數增加對偵測時間與MIC的影響----------------------61
3.4電容量量測法作陽性血瓶(positive blood cultures)中
酵母菌藥物敏感性試驗-------------------------64
3.4.1陽性血瓶中酵母菌之測定---------------------------64
3.4.2陽性血瓶中酵母菌的菌種分布-----------------------64
3.4.3陽性血瓶電容量測法之MIC與傳統法的吻合度----------66
3.4.4培養時間對電容量法直接量測MIC之影響--------------67
3.5陽性血瓶直接進行藥物感受性試驗---------------------68
3.5.1受檢陽性血瓶菌種分佈-----------------------------68
3.5.2區分點直接藥物敏感性試驗與傳統方法之比較---------69
3.6以Etest直接進行陽性血瓶抗真菌藥物的MIC測定---------70
3.6.1 Etest與傳統方法比較（純菌之測試）---------------70
3.6.2 Etest直接接種法與傳統方法的比較-----------------71
第四章 結論-------------------------------------------73
4.1以電容量測法檢測抗真菌藥物對純菌最低抑菌濃度響應及吻合度的探討----------------------------------------------------73
4.2陽性血瓶培養液直接進行藥物敏感性試驗測試-----------73
4.3 Etest檢測-----------------------------------------74
參考文獻-----------------------------------------------75

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