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研究生：	阮家豪 Ruan, Jia-Hao
論文名稱：	一種預估嚴重登革熱風險的快速檢測法之開發 Development of a Rapid Test for Risk Prediction of Severe Dengue
指導教授：	張憲彰 Chang, Hsien-Chang
學位類別：	碩士 Master
系所名稱：	工學院 - 生物醫學工程學系 Department of BioMedical Engineering
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	54
中文關鍵詞：	嚴重登革熱、乳膠微珠凝集、抗體表面修飾、影像處理、非結構蛋白1 、人類凝血酶、快速生物標記篩檢
外文關鍵詞：	Severe dengue, latex agglutination, antibody-modified surface, image processing, non-structural protein 1, human prothrombin, rapid biomarker screening
相關次數：	點閱：111 下載：0
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登革熱是一種藉由蚊媒傳播之病毒傳染病，目前已在全球熱帶及亞熱帶造成嚴重疫情，並導致昂貴的社會成本支出。登革熱初期的臨床症狀如發燒等容易和常見流感等病毒感染症混淆，但卻可能在數日後發生出血或休克等致命併發症，因此強化高精準登革熱診斷之快速篩檢尤其能夠預測重症是有迫切需求的。目前臨床上大多採用登革病毒的非結構蛋白1 (NS1)作為快篩之標的，但受限於患者個體免疫反應的不同、受檢測時病程的差異，以及現有技術的限制等因素而造成偽陰性結果。目前臨床大多僅能藉由追蹤患者受感染後的特徵預測病情的嚴重性，若能提供高專一性、便宜的登革熱預測性檢測，將可滿足臨床上對於病情監控的需求。本研究整合乳膠微珠凝集方法及影像處理演算法，針對嚴重登革熱的特異性生物標記—NS1-凝血酶複合體 (NST)開發了一套可以在20 min內針對患者發展成嚴重登革的風險預測平臺。透過在1 μm及0.3 μm直徑的乳膠微珠表面固定化針對NS1-凝血酶複合體 (NST)抗原之特異性抗體，令抗體微珠探針與患者血清稀釋液在玻片上反應，當樣本中之NST濃度與微珠濃度具一特定比例關係時，就可觀察到乳膠微珠聚集的現象。完成反應後的玻片再利用顯微影像擷取影像，並使用開發之影像處理算法進行自動化分析，搭配統計學方法即可快速得出樣本中的NST抗原濃度對應之嚴重登革熱風險等級。本檢測平台目前在包括對照組在內的36個樣本中，預測的靈敏度為81.8%，特異性為85.7%。利用本方法將可進一步縮短如傳統生物標記快速篩檢所需的時間，也可改進成僅憑裸眼判讀的微珠凝集法，就可得到較粗略之及時結果。希冀未來可拓展至臨床檢測上，提供快速之重要指標來監控預將惡化轉為登革熱重症(登革出血熱(DHF)或登革熱休克症候群(DSS))等的可能性。

Dengue is a mosquito-borne viral infection that has caused serious epidemics in tropical and subtropical zones around the world. The initial clinical symptoms of dengue fever, such as fever, can be easily confused with common viral infections such as influenza, but fatal complications such as hemorrhage or shock can occur days later, so there is an urgent need to strengthen rapid screening for severe dengue. Currently, most clinical trials use the non-structural protein 1 (NS1) of the dengue virus as a quick screen for patients and can only predict the severity of the disease by following the characteristics of patients after infection. Therefore, a highly specific and inexpensive predictive test for dengue would meet the clinical need for monitoring the disease. This study integrates latex bead agglutination methods and image processing algorithms to develop a platform for predicting a patient's risk of developing severe dengue within 20 min by targeting NS1-thrombin complex (NST), a specific biomarker for severe dengue. Antibodies specific for the NST antigen were immobilized on the surface of latex beads of 1 and 0.3 μm in diameter, and the antibodies were allowed to react with the patient's serum diluent on a slide. An image processing algorithm was developed to automate the analysis of the completed slides and, together with statistical methods, to rapidly determine the level of risk of severe dengue fever corresponding to the concentration of NST antigen in the sample. With this platform, it will be possible to further reduce the time required for rapid screening of traditional biomarkers and avoid the interpretation errors caused by traditional microbead agglutination methods. The platform currently has a prediction sensitivity of 81.8% and specificity of 85.7% in 36 samples including controls. Given the success of the platform, it will hopefully provide a rapid diagnostic basis for severe dengue.

Abstract I
中文摘要 II
誌謝 V
List of Figures VI
List of Tables IX
Chapter 1 Introduction 1
1 Background 1
1.1 Dengue 1
1.2 Severe Dengue 2
1.3 Specific Biomarker for Severe Dengue 2
2 Current Methods for Dengue Diagnosis 4
2.1 Rapid Detection Method Based on Lateral Flow 6
2.2 Enzyme-linked Immunosorbent Assay 7
2.3 Nucleic acid amplification-based assay 8
2.4 Unmet Need of Severe Dengue Diagnosis 9
3 Motivation and Aims 9
4 Research Framework 10
Chapter 2 Materials and Methods 11
1 Materials 11
2 Latex Agglutination 12
2.1 Latex Agglutination Assay 12
2.2 Hook Effect 13
3 Optimization of Antigen-antibody Response 14
4 Latex Beads Functionalization 16
5 Agglutination by Using Two Sizes of Beads 17
6 Image Process Method Version 1: Average Area 18
7 Particle Size Distribution after Agglutination 20
8 Precipitation Curve of Serial Diluted Samples 21
9 Establishment of NS1 Calibration Curve 22
10 Image Process Method Version 2: Particle Density 23
11 NST Agglutination with Particle Density Method 24
12 Receiver Operating Characteristic Curves for Severe Dengue Risk Classification 25
13 NST Agglutination in Serum/Plasma 27
Chapter 3 Results and Discussion 28
1 Optimization of Antigen-antibody Response 28
2 Latex Beads Functionalization 31
3 Agglutination by Using Two Size of Beads 32
4 Particle Size Distribution after Agglutination 34
5 Precipitation Curve of Serially Diluted Samples 35
6 Establishment of NS1 Calibration Curve 36
7 Concentration Prediction with NS1 Standard Sample 37
8 Method Comparison: Average Area vs. Particle Density 38
9 NST Agglutination with Particle Density Method 39
10 ROC Curves to find Severe Dengue Binning Threshold 41
11 Risk Prediction for Severe Dengue with NS1/NST 44
12 Interferences Test of Immunoassay with NST-Spiked Plasma/Serum 46
13 Risk Prediction with NST Spiking Sample 48
Chapter 4 Conclusion and Prospects 50
Reference 51
Personal Information 54
                                    

1. Anderson, J.R. and R. Rico-Hesse, Aedes aegypti vectorial capacity is determined by the infecting genotype of dengue virus. Am J Trop Med Hyg, 2006. 75(5): p. 886-892.
2. Brady, O.J., et al., Refining the Global Spatial Limits of Dengue Virus Transmission by Evidence-Based Consensus. PLoS Negl Trop Dis, 2012. 6(8): p. e1760.
3. Carrington, L.B., et al., Fluctuations at a Low Mean Temperature Accelerate Dengue Virus Transmission by Aedes aegypti. PLoS Negl Trop Dis, 2013. 7(4): p. e2190.
4. Watts, D.M., et al., Effect of temperature on the vector efficiency of Aedes aegypti for dengue 2 virus. Am J Trop Med Hyg, 1987. 36(1): p. 143-152.
5. Ho, T.-S., et al., Clinical and laboratory predictive markers for acute dengue infection. J Biomed Sci, 2013. 20(1): p. 75.
6. Lambrechts, L., et al., Impact of daily temperature fluctuations on dengue virus transmission by Aedes aegypti. Proc Natl Acad Sci U S A, 2011. 108(18): p. 7460-7465.
7. Bhatt, S., et al., The global distribution and burden of dengue. Nature, 2013. 496(7446): p. 504-507.
8. Scaturro, P., et al., Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins. PLOS Pathog, 2015. 11(11): p. e1005277.
9. Flamand, M., et al., Dengue virus type 1 nonstructural glycoprotein NS1 is secreted from mammalian cells as a soluble hexamer in a glycosylation-dependent fashion. J Virol, 1999. 73(7): p. 6104-6110.
10. Chen, M.-C., et al., Deletion of the C-Terminal Region of Dengue Virus Nonstructural Protein 1 (NS1) Abolishes Anti-NS1-Mediated Platelet Dysfunction and Bleeding Tendency. J Immunol, 2009. 183(3): p. 1797.
11. Lai, Y.-C., et al., Antibodies Against Modified NS1 Wing Domain Peptide Protect Against Dengue Virus Infection. Sci Rep, 2017. 7(1): p. 6975.
12. Lin, Y.-S., et al., Molecular mimicry between virus and host and its implications for dengue disease pathogenesis. Exp Biol Med, 2011. 236(5): p. 515-523.
13. Libraty, D.H., et al., High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever. J Infect Dis, 2002. 186(8): p. 1165-1168.
14. Cheng, H.J., et al., Correlation between serum levels of anti-endothelial cell autoantigen and anti-dengue virus nonstructural protein 1 antibodies in dengue patients. Am J Trop Med Hyg, 2015. 92(5): p. 989-995.
15. Lin, S.-W., et al., Dengue virus nonstructural protein NS1 binds to prothrombin/thrombin and inhibits prothrombin activation. J Infect, 2012. 64(3): p. 325-334.
16. World Health, O., Dengue guidelines for diagnosis, treatment, prevention and control : new edition. 2009, World Health Organization: Geneva.
17. Waggoner, J.J., et al., Viremia and Clinical Presentation in Nicaraguan Patients Infected With Zika Virus, Chikungunya Virus, and Dengue Virus. Clin Infect Dis, 2016. 63(12): p. 1584-1590.
18. Tjaden, N.B., et al., Extrinsic Incubation Period of Dengue: Knowledge, Backlog, and Applications of Temperature Dependence. PLoS Negl Trop Dis, 2013. 7(6): p. e2207.
19. Avirutnan, P., et al., Vascular leakage in severe dengue virus infections: a potential role for the nonstructural viral protein NS1 and complement. J Infect Dis, 2006. 193(8): p. 1078-1088.
20. Halstead, S.B., et al., Intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes. Lancet Infect Dis, 2010. 10(10): p. 712-22.
21. Srikiatkhachorn, A., et al., Virus-induced decline in soluble vascular endothelial growth receptor 2 is associated with plasma leakage in dengue hemorrhagic Fever. J Virol, 2007. 81(4): p. 1592-600.
22. Bouzid, D., et al., Rapid diagnostic tests for infectious diseases in the emergency department. Clin Microbiol Infect, 2021. 27(2): p. 182-191.
23. Hansson, J., et al., Synthetic microfluidic paper: high surface area and high porosity polymer micropillar arrays. Lab Chip, 2016. 16(2): p. 298-304.
24. Koczula, Katarzyna M. and A. Gallotta, Lateral flow assays. Essays Biochem, 2016. 60(1): p. 111-120.
25. Weiland, G., [The enzyme-linked immunosorbent assay (ELISA)--a new serodiagnostic method for the detection of parasitic infections (author's transl)]. MMW Munch Med Wochenschr, 1978. 120(44): p. 1457-1460.
26. Kragstrup, T.W., et al., A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients. Springerplus, 2013. 2(1): p. 263.
27. Lancefield, R.C., The antigenic complex of streptococcus haemolyticus : I. Demonstration of a type-specific substance in extracts of streptococcus haemolyticus. J Exp Med, 1928. 47(1): p. 91-103.
28. Ward, G., et al., The investigation of interferences in immunoassay. Clinical Biochemistry, 2017. 50(18): p. 1306-1311.
29. Hirst, G.K., The quantitative determination of influenza virus and antibodies by means of red cell agglutination. J Exp Med, 1942. 75(1): p. 49-64.

2026-08-17公開

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