2014-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/658924摘要：雖然微生物學及分子生物學的診斷工具及技術不斷的在進步，然而早期診斷感染致病原，以幫助臨床醫師決定正確的抗微生物藥物及治療決策，仍然是臨床上一項困難的挑戰，這不僅是由於檢驗室在細菌或病毒培養鑑定上的困難及所必需的檢驗操作時間，也在於臨床上致病微生物的多樣性，使得利用傳統的微生物檢驗或使用僅具特定菌種檢測力的分子生物學方法來診斷感染致病原，在時效性及敏感度上都有著極大的限制，形成臨床實用上的困難，進而造成治療藥物或必要介入措施的延遲，影響病患治療預後至鉅。因此，要發展新型快速且準確的微生物診斷技術，必須跳脫傳統微生物及分子生物學的窠臼，而從另一種同樣具專一性的宿主免疫反應這樣不同的面向來思考。 學理上，隨著不同的致病菌入侵體內造成感染，人體的免疫系統會針對該種致病菌產生具專一性的免疫細胞反應，其中 T細胞更藉由不同的蛋白結構上千萬種的組合，形成可以跟外來物質結合的接受器(receptor)以辨識各種侵入人體內不同的致病原，進而引起一連串的免疫反應以抵抗消除這些外來的致病原。正因 T細胞扮演著重要的免疫辨識、反應及調整的關鍵角色，所以偵測體內 T細胞因為特定感染所產生的專一性免疫力造成整體免疫組庫的變化，就成為提供臨床診斷致病細菌種類的可能途徑。雖然分析 T細胞中所可能具有的上千萬種免疫組庫(immune repertoire)在過去對免疫及分子生物學家形成研究上的困難，但現在利用arm-PCR(amplicon-rescued multiplex PCR)，配合高通量定序(high throughput sequencing)，現在已經可以針對 T細胞受器β鍊(T cell receptor β chain , TCRβ)的所有免疫組合型態、個別相對強度、及多樣性進行分析。 鑒於臨床上對於致病菌診斷上的困難與延遲對病患預後的影響，因此本研究的主要目的，在於藉由分析金黃色葡萄球菌、克列白氏菌、及沙門氏菌菌血症病患在感染急性期的 T細胞免疫組庫的型態及相對強度，找出人體免疫系統對上述致病菌所產生的特定免疫反應型態(bacteria-specific immune repertoire pattern)，作為提供感染症早期診斷的依據。同時本研究將進一步分析這些特定免疫組庫的多樣性時序變化，是否可以用來預測病患預後以及是否出現深部組織持續性感染，以提供將來臨床醫師早期診斷與正確治療的參考。最後，本研究將配合臨床菌株的分子分型分析，探討細菌菌株、臨床感染部位，及宿主免疫力的交互作用，以釐清影響病患預後的相關因子。<br> Abstract: There are dramatic advances in the microbiological and molecular diagnostic techniques in recent decades. However, early identification of causative micro-organism of an infection remains a diagnostic challenge. It has also been observed that delay in clinical and microbiological diagnosis result in inappropriate empirical antimicrobial therapy and management decision making, therefore impacts the treatment outcomes of patients with sepsis. It is there imperative to develop a novel laboratory tool for early microbiological diagnosis and to improve clinical outcomes of patients. Theoretically, T-cell plays a critical role in human immunity. T-cell can produce pathogen-specific immunity by adjusting the immune repertoire status when facing exotic micro-organisms. Therefore, it is plausible to diagnose potential causative micro-organism by detecting the pathogen-specific changes in pattern, intensity, and diversity of T-cell immune repertoire during an active infection. Though the diversity and computation difficulty impeded the analysis of T-cell immune repertoire previously the development of new arm-polymerase chain reaction and high throughput sequencing method have overcome the technique obstacle in this field. In our proposal, we plan to analyze the T-cell immune repertoire pattern among patients with Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, and Salmonella bacteremia. The intensity and pattern of study patients’ T-cell immune repertoire will be identified and compared with those of healthy population to establish the pathogen-specific immune patterns of the four target study bacteria. Furthermore, we will determine the correlation between the change in T-cell immune repertoire diversity during different stages of infection and clinical outcomes of bacteremia patients. Finally, the interaction between bacteria strain, infection site, and host immunity will be studied to clarify the independent influencing factors that contributes patient mortality.T細胞免疫組庫arm-聚合脢連鎖反應高通量定序分子分型細菌感染早期診斷預後T-cell immune repertoirearm-poly chain reactionhigh throughput sequencingmolecular analysisbacterial infectionearly diagnosisprognosis.