(100IDP008-3)Rapid Diagnosis of Active Tuberculosis from Sputum, Blood, and Urine Specimens (I)

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Project title/計畫英文名

Project Number/計畫編號

NSC100-2325-B002-010

Translated Name/計畫中文名

可攜式微型化SPR-LAMP方法於肺結核菌快速診斷之系統研發---(子計畫三)由痰、血液、及尿液檢體快速診斷結核病(I)

Project Principal Investigator/計畫主持人

JANN-YUAN WANG

Funding Organization

National Science and Technology Council

Co-Investigator(s)/共同執行人

李麗娜

Website

link

Start date/計畫起

01-05-2011

Expected Completion/計畫迄

12-04-2012

摘要：結核病仍是目前全世界最重要的感染症。2008 年，台灣地區結核病新個案有14,265 人，發生率仍然高達每十萬人口中有62 人。想要成功地控制結核病，必須能夠早期診斷、早期治療，以避免結核菌進一步散播。然而，使用最普遍的抗酸性染色對於診斷結核病不但敏感度低，還需要花費相當多的人力和時間，同時沒有辦法區分結核菌與非典型分枝桿菌。特別是在非典型分枝桿菌盛行率很高的地區，這個問題更顯嚴重。分枝桿菌的培養雖然有很高的敏感度與特異度，但就算用液態培養設備，也必須要10天以上的時間。為了彌補這些缺點，利用核酸增幅的快速診斷試劑因應而生。然而，由於低敏感度（針對抗酸性染色陰性之檢體，敏感度大約0.66）和高成本（一次測試約新台幣1,000~1,500 元），臨床上核酸增幅試劑對於結核病診斷上的應用仍然受到相當大的限制。此外，隨著抗藥性結核菌的問題日益嚴重，為了即早診斷以避免抗藥性的進一步傳播，快速診斷結核菌的抗藥性，也變成是十分重要的議題。有鑑於臨床上的迫切需要，我們藉由整合環化促成恆溫增幅反應以及微型化有機發光二極體表面電漿共振生物檢測系統建置高效能、低成本的快速檢驗平台，希望能夠利用各種臨床檢體快速診斷結核病及其抗藥性。我們將於 2011/5/1 至2014/4/30 期間，在臺大醫院，前瞻性地納入200 位疑似肺結核之成人個案並收集痰、血液、尿液檢體，並萃取出其中的DNA。再利用我們研究團隊所開發之環化促成恆溫增幅反應及微型化有機發光二極體表面電漿共振生物檢測系統，檢測臨床檢體中有無結核菌基因序列或抗藥基因存在。檢測結果的準確性將根據病人臨床資料（包括檢體耐酸性染色、分枝桿菌培養、病理檢查、影像學檢查結果以及抗結核藥物治療反應）綜合判斷是否為肺結核個案，並與現行臨床上最廣為使用之核酸增幅試劑檢驗結果比對（Roche Cobas TaqMan MTB test）。研究目的：1) 開發便宜、便利、準確度高的結核病快速診斷試劑，經由臨床試驗證實其準確性。2) 開發適用各種臨床檢體（痰、血液、尿液、體液）的結核病快速診斷試劑3) 快速診斷結核菌之抗藥性。
Abstract: Tuberculosis remains the most important infectious disease in the world. In 2008,a total of 14,265 new cases were reported in Taiwan and the incidence was 62 per100,000 populations. Early diagnosis and early treatment to prevent furthertransmission are the key steps for tuberculosis control. However, early diagnosis isnot easy. Acid-fast smear, the most popular rapid diagnostic test for tuberculosis, hasa low sensitivity in comparison with mycobacterial culture and is labor-dependent andtime consuming. In addition, acid-fast smear cannot differentiate betweennontuberculous mycobacteria and Mycobacterium tuberculosis. The situation wouldbe more serious in area of high incidence of NTM disease. Mycobacterial culture,though having a high sensitivity and specificity, requires at least 10 days before apositive result can be available. Therefore, rapid diagnostic assays using nucleicamplification test are developed. However because of low sensitivity (around 0.66 forsmear-negative respiratory samples) and high cost (NTD 1,000~1,500 per test),clinical use of nucleic acid amplification test for rapid diagnosis of tuberculosis is stilllimited. In addition, as the incidence of drug-resistant tuberculosis increases, earlydiagnosis of drug resistance becomes an important clinical issue.Due to the urgent need, we try to develop a high-throughput, low-costrapidly diagnostic assay incorporating loop mediated isothermal amplication(LAMP) and organic light emitting diodes surface plasmon resonance(OLED-SPR) micro bio-sensor (OLED-SPR) and to test its performance(detecting M. tuberculosis DNA and resistant genes) using clinical samples.From May 01, 2011 to April 30, 2014, a total of 200 clinical suspects ofpulmonary tuberculosis in National Taiwan University Hospital will be enrolled andasked to collect sputum, blood and urine samples. After DNA extraction and LAMP, M.tuberculosis or drug resistance gene in clinical samples will be detected by using theOLED-SPR our team developed and compared with the results of the most popularnucleic acid amplification test for tuberculosis (Roche Cobas TaqMan MTB test). Thediagnosis of tuberculosis will be judged based on all clinical data (including results ofacid-fast smear, mycobacterial culture, histology examination, radiographic findings,and response of anti-tuberculous treatment).Specific aims:1) To develop cheap, portable, and accurate test for rapid diagnosis of tuberculosisand to verify its performance on clinical samples2) To develop a rapid diagnostic assay that is able to apply on many clinical samples(sputum, blood, and urine samples)3) Rapid diagnosis of drug resistance

Keyword(s)

結核菌
快速診斷
環化促成恆溫增幅反應
有機發光二極體
表面電漿共振
細胞凋零
decoy receptor 3
prostaglandin E2
lipoxin

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(100IDP008-3)Rapid Diagnosis of Active Tuberculosis from Sputum, Blood, and Urine Specimens (I)

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