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題名：感染病原體及宿主免疫反應即時檢測方法開發及臨床驗證
作者：陳正翰
作者(外文)：Chen, Cheng-Han
校院名稱：國立清華大學
系所名稱：跨院國際博士班學位學程
指導教授：鄭兆珉
學位類別：博士
出版日期：2022
主題關鍵詞：敗血症；即時檢測；側向流體免疫分析法；IL-6快速篩檢系統；細胞激素陣列分析；Sepsis；bacteria detection；Interleukin-6 rapid diagnostic system；Lateral flow immunoassay；point-of care testing；Cytokines array
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Sepsis remains a considerable threat to health loss and socio-economic burden in the contemporary healthcare system. Sepsis mortality is significantly rising if delayed diagnosis and inappropriate treatment and management. However, the variety of patients' clinical presentations and the labor-intensive laboratory examinations impede the diagnosis of sepsis. Therefore, it is essential to recognize the critical information of septic patients early and effectively. Nowadays, the crucial information about controlling sepsis might be separated into two major issues: pathogen identification and host immune response. This doctoral project aims to develop point-of-care (POC) devices assisting sepsis management and further evaluate the potential effectiveness of these POC devices in clinical samples.
Prompt recognition of the presence of microorganisms is critical for controlling infectious diseases. Standard culture methods, however, are time-consuming, inefficient, and laborious. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-phenazine methosulfate (MTT-PMS) assay has been utilized for screening living microbes. By adding NaOH and Tris-EDTA, the process of the modified assay can be expedited (within 15 min) and quickly detect common living microbes. The results of the MTT-PMS assay can be assessed colorimetrically and semi-quantitatively via the ELISA reader. The assay had a detection limit of approximately 104 CFU/mL in buffered system. In the clinical efficacy evaluation, the MTT-PMS assay showed the negative predictive value for detecting living microbes at the concentration of 105 CFU/mL was approximately 80 percent in urine samples but was 100 percent when the redox interference of abnormal blood in urine was excluded. Thus, the MTT-PMS assay might provide a conceivable screening result costing only USD 1 per test. Given the features of rapid results, easy to use, and low expenditure, the MTT-PMS assay may be a potential tool for microorganism detection.
Interleukin-6, on the other hand, is a cytokine that will be released into the bloodstream in response to sepsis. Previous studies had demonstrated the elevation of Interleukin-6 (IL-6) indicated a higher possibility of poor outcome. However, the conventional measurement of IL-6 of the enzyme-linked immunosorbent assay (ELISA), which comprises several steps, and is time consuming and laborious, impedes the IL-6 measurement in clinical utilization. The IL-6 rapid diagnostic system combined with a lateral flow immunoassay-based IL-6 test strip and a spectrum-based optical reader is a novel tool developed for rapid and continuous bedside monitoring of serum IL-6. The prospective observational study demonstrated the efficacy of serum IL-6 detection platform in predicting respiratory failure of the elderly patients with pneumonia. In consecutive measurement, decreased serum IL-6 concentrations within 24 h after admission indicated a lower risk of developing respiratory failure (ROC curve=0.696, p=0.072) Sequential IL-6 measurements with the IL-6 rapid diagnostic system might be useful in early clinical risk assessment and severity stratification in elderly patients with pneumonia. The IL-6 rapid diagnosis system is a potential point-of-care diagnostic device for continuous serum IL-6 monitoring. Besides the IL-6 measurement, a sub-analysis study with cytokine array demonstrated the relationship among significant decreased serum IL-1β after admission and the development of respiratory failure in later admission courses. These findings could be further integrated the other cytokines into the new POC devices development.
Thus, this doctoral project discovered the potential clinical application of the point-of-care devices with the MTT-PMS assay and the IL-6 rapid diagnostic system. These POC devices for rapid sepsis management could be further optimized by incorporating machine learning and the internet of things (IoT). The quality of care for sepsis can be improved with the rapid, ease-to-use, and disposable POC devices for bacterial screening and host immune response evaluation.
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