結合奈米脂粒與抗體微陣列晶片的高通量快速檢測系統之發展並應用於婦女子宮頸炎病因之診斷與研究

A chip-based multiplexed immunoassay system using liposomal nanovesicles and its application on the detection of pathogens causing female cervicovaginitis

蘇文祥(Wen-Hsiang Su)

指導教授 ： 陳健生

國立中央大學/理學院/系統生物與生物資訊研究所/博士(2012年)

摘要

中文摘要 (Chinese Abstract)
研究目的：在婦產門診業務中，最常遇到的疾病就是女性的子宮頸炎或者陰道炎，尤其是在接受常規抹片檢查逐漸被婦女同胞認同而逐漸普遍的今天，抹片報告「發炎」一直是困擾著病患也困擾著醫師的一大難題，如果有更快速而且有效率的檢驗方法，可以告訴我們到底發炎是由哪些致病體所引貣的？這個發炎會不會發展成為癌症？是否需要積極的治療以預防更嚴重的感染，如子宮炎、輸卵管炎、骨盆腔炎、甚至卵巢膿瘍，一些嚴重的影響婦女健康的炎症？我們將利用本實驗室在高通量生物感測的專長，在抗體微陣列晶片上建立奈米脂粒的病原體抗體反應訊號放大與偵測系統。該系統建立之後，我們將利用各病原體菌株來進行該偵測系統的效度測試。等達到一定的敏感度和特異性之後，接著將申請臨床試驗，蒐集參與者陰道分泌物檢體 (只頇數十個 microliter)進行分組測試，以確定本系統偵測與鑑別診斷的能力。針對這樣的疾病和工具，我們集合系統生物與生物資訊研究所的微晶片實驗室提出了、台北榮民總醫院的資源配合中央大學統計研究所的專長，在民國一百年度申請到了榮總台灣聯合大學合作研究整合型計畫，雖然對於我們想要用數學模式模擬正常與有症狀的陰道微生物菌落組成，仍有待努力蒐集更多的實驗室和臨床數據，我們依然順利的完成了該計畫，並且發表了兩篇論文並且完成第三篇論文投稿。
資料來源與研究方法：經過文獻查閱與網路搜尋，並且配合本身條件與有限經費，我們在多種引貣陰道子宮頸炎的主要病原菌中選擇了五種作為標的，包括披衣菌、淋病雙球菌、大腸桿菌、乙型鏈球菌和白色念珠菌的抗體被買來點在玻片上。一個奈米微脂粒訊號放大與偵測系統被建立貣來並且設計實驗來驗證該系統的正常運作。然後在實驗室裡以抗原與病原菌測試該系統確實可以偵測到晶片上的抗體抗原反應。另外最後經由婦產科門診收集病患的陰道與子宮頸分泌物進行病原菌的檢測並且以加回的方法驗證本系統的確可以偵測到檢體中的白色念珠球菌。在將來我們將使用數學比例勝算模組來做運算基礎，嘗試鑑別嚴重陰道炎的微生物組成。結果是否有意義將經由配對t 試驗來檢驗，p<0.05 為有意義。
研究結果：在整個文獻查詢和臨床應用可能性的查證之中，我們針對淋病感染的診斷工具做了一番回顧，在比較過優缺點和是否能在門診及時應用的可能性後，提出了最好的方法還在建構中的結論。本系統也被初步證實可以在實驗室中可偵測微克級披衣菌抗原，並且呈現濃度與訊號強度有正相關，針對與以往診斷披衣菌感染的診斷工具的不同處和本系統在敏感度提升的可能性，我們又做了一番整理與發表。最後對於其他病原菌，這個系統在實驗室檢體中的偵測能力被證實，p 值小於0.01。特別在白色念珠菌的偵測敏感度高達每毫升十萬菌落數的水準，而在臨床檢體中，本系統對於加回該菌的敏感度與專一性也都接近百分之百。而且一般處理時間大概只需要兩百分鐘，而所需要的偵測工具只是一般的實驗室器材和一台螢光數據讀取儀器。
結論：，本偵測系統提供了相對經濟、簡單、高通量、快速、靈敏、奈米級的多重病原體偵測工具，至少對白色念珠菌而言是如此。這個領域的進步，終將可以幫助臨床醫護人員快速、經濟而準確的鑑別診斷在陰道中可能引貣子宮頸炎的不同菌種。

關鍵字

奈米脂粒 ； 陰道子宮頸炎 ； 微晶片 ； 脂肪小體 ； 快速診斷

並列摘要

English Abstract
Backgrounds: Cervicovaginitis is a common infection in women. Ignorance of this disease may place a heavy burden on female and neonatal health due to occurrence of severe sequelae such as female infertility, ectopic pregnancy, chronic pelvic pain, and neonatal infections. Prompt and appropriate antibiotic treatment can cure infection and avoid complications. However, adequate treatment is not easy, since many factors can interfere with an early and rapid identification of various pathogens from complicated mixed micro-flora of the vagina and cervix. Among these factors, a non-user-friendly rapid detection system seems the most important. A microarray immunoassay chip was fabricated for binding of pathogens causing cervicovaginitis and a signal amplifying system using liposomal nanovesicles was used for detection. Laboratory and clinical samples were tested with some promising results. We collected the source from Veteran General Hospital in Taipei and Hit Laboratory in the department of Systems Biology and Bio-informatics. With expert help from Statistic school of National Central University, our project was accepted and granted by the aiwan Joint Research Program (VGHUST99-G4-3) from Jan. 1st to Dec. 31st, 2010. Although the attempt to simulate a normal and symptomatic micro-flora in the vagina by mathematic model demanded more work from laboratory and clinic setting. We still finished the project with limited resources and published two SCI manuscripts and submitted our third one.
Material and methods: After literature review and data mining on the internet, five pathogens were chosen based on our limited culture ability and financial support. Well established capture antibodies for specific pathogens causing cervicovaginitis, including Streptococcus B, Chlamydia trachomatis , Neisseria gonorrhoeae , Escherichia coli, and anti Candida albicans were purchased to be printed on a Chip. Signal amplifying system using liposomal nanovesicles was established and validated. Various pathogens and antigens were collected for validation of this detecting system on chips. Cervical and vaginal samples were obtained from an ordinary gynecologic clinic for pathogens detection. Spiking of candida in the samples were used to testify the usefulness of this detection system. We will use the proportional odds models to make inferences regarding which pathogen tends to induce more severe infection if more positive data available. The difference between positive and negative readings was evaluated by paired t test.
Results: During our literature review and data mining for the application of our tool in the diagnosis of specific infections, we focused on the gonococcal infection first. After comparison with other tools, we argued the best method for the diagnosis of gonorrhea infection is still under construction especially regarding the qualities as a Point-of-Care tool. Furthermore, our system was proved to be sensitive in detecting Chlamydia antigen to a level of micro-gram. A positive proportion relationship between antigen concentrations and signal intensity was also demonstrated. Another review about the possibility of our system and comparison with other tools for diagnosis of Chlamydia infection was completed and published. As for other pathogens, the power of this system was validated (p<0.01). The detection sensitivity of candida of this system reach the level of 100,000 CFU/ml in a vaginal sample. In the study of clinical samples, the sensitivity and specificity of this tool in the candida spike test reach near 100% sensitivity and specificity. Moreover, the average processing time is around 200 minutes and the required apparatus is ordinary laboratory equipments with a fluorescent reading machine.
Conclusions: Microarray chip is a relatively rapid, easy, inexpensive and sensitive tool to detect many pathogens, at least for Candida, in a one-time vaginal sampling process in our Laboratory. Eventually, advances in related laboratory techniques will satisfy our needs to detect pathogens in vaginal and cervical samples economically and instantly.

並列關鍵字

cervicovaginitis ； rapid diagnosis ； microchip ； liposome ； nanovesicle

參考文獻

1. Pellati, D., et al., Genital tract infections and infertility. Eur J Obstet Gynecol Reprod Biol, 2008. 140(1): p. 3-11.

2. Heinonen, P.K. and M. Leinonen, Fecundity and morbidity following acute pelvic inflammatory disease treated with doxycycline and metronidazole. Arch Gynecol Obstet, 2003. 268(4): p. 284-8.

3. Doxanakis, A., et al., Missing pelvic inflammatory disease? Substantial differences in the rate at which doctors diagnose PID. Sex Transm Infect, 2008. 84(7): p. 518-23.

4. Ness, R.B., et al., A cluster analysis of bacterial vaginosis-associated microflora and pelvic inflammatory disease. Am J Epidemiol, 2005. 162(6): p. 585-90.

5. Heinonen, P.K. and A. Miettinen, Laparoscopic study on the microbiology and severity of acute pelvic inflammatory disease. Eur J Obstet Gynecol Reprod Biol, 1994. 57(2): p. 85-9.