免疫流行病學：2006年台灣地區登革熱/登革出血熱病人病毒量、免疫反應、臨床嚴重度及流行病學特徵之關係

Immunoepidemiology: The Relationship among Epidemiological Characteristics, Clinical Severity, Viral Load and Immune Responses in Patients of Dengue Fever versus Dengue Hemorrhagic Fever in Taiwan, 2006

李宜軒(Yi-Shiuan Li)

指導教授 ： 金傳春

國立臺灣大學/公共衛生學院/流行病學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.02772

摘要

登革出血熱(Dengue hemorrhagic fever, DHF)與登革熱(Dengue fever, DF)是由登革病毒(Dengue virus, DENV)所引起的急性蟲媒傳染病，其流行中滋生嚴重病例之致病機制至今仍無法完全解釋。本研究包含兩大部分：(一) 2006年通報至台灣疾管局之全國登革確定病例進行流行病學分析，及(二)登革病毒免疫流行病學深入探究，由定點醫師收集2006年高屏地區登革流行中發病而願參與者之血液，利用同一人不同發病時點的樣本進行病例對照研究，以第三型登革病毒即時定量反轉錄聚合酶連鎖反應[One-step TaqMan-based real-time reverse transcriptase polymerase chain reaction (RT-PCR)]及液相多重蛋白定量技術(BD™ Cytometric Bead Array, CBA)，分別偵測血清中病毒量及各種細胞激素/化學激素(Cytokines/Chemokines)之濃度，是第一次整合流行病學、免疫學、病毒學及臨床表徵四角度之貫穿數據分析，欲探討同一病人不同發病時程（急性期、退燒期、恢復期)之病毒量、Th1/Th2免疫/發炎反應的細胞激素/化學激素及其交互作用是否與健康對照組不同；又同一流行區隨時間之拉長與傳播密度之增高是否其臨床症狀呈現更趨嚴重度之關聯性

關鍵字

登革熱/登革出血熱 ； 病毒量與致病 ； 免疫致病機轉 ； 細胞激素/化學激素 ； 流行嚴重機轉 ； 蟲媒傳染病 ； 台灣

並列摘要

Mechanisms of dengue hemorrhagic fever (DHF) caused by dengue virus (DENV) have attracted many interests. To our knowledge, this is the first study that integrated epidemiology, clinical manifestations, immunology and virology to investigate the changing patterns of viral and host immune responses along the epidemic of dengue /DHF that might be associated with the increasing severity of DHF cases. This study involved two parts: (1) epidemiological analysis of the 2006 epidemic of dengue /DHF in southern Taiwan, and (2) immuno-epidemiological case-control study on the interplays between viral and human host factors at three different stages of disease process of patients (acute, defervescence, and convalescent) and in various epidemic time periods (early, middle and late) and areas (high or low transmission intensity). Blood samples of DF/DHF patients in southern Taiwan from June to December in 2006 collected by our sentinel physicians through active surveillance plus age-/gender-matched healthy controls were used to measure the viral load and the concentrations of cytokines and chemokines by one-step dengue virus serotype 3 (DENV-3) specific TaqMan-based real-time reverse transcriptase polymerase chain reaction (RT-PCR) and BD™ Cytometric Bead Array (CBA) respectively. Through comparison in quantitative measurements of Th1/Th2 and inflammatory cytokines and chemokines and their association with viral load and clinical manifestations/severity plus subsequent stratification and multivariate epidemiological data analysis, we can elucidate immuno-pathogenesis mechanism(s) of DHF at both individual and population levels under the unique epidemiological characteristics of dengue in Taiwan.
Epidemiological analysis of the 1076 official confirmed dengue cases [1057 dengue fever (DF) cases and 19 DHF cases] reported to Taiwan-CDC showed that the overall incidence rate was 0.822 per million and case fatality rate of DHF was 21.05% (4/19). DENV-3 was the dominant serotype (81.7%, 389/476). Most indigenous dengue cases occurred in Kaohsiung and Pingtung (98.14%, 949/967) and its incidence rate was 2.670 per million. The age older than 40 was one of the risk factors contributing to DF (56.5%, 597/1057) and DHF (63.2%, 12/19) (p=0.014) but the percentage of DHF cases in children ≦17 year-old in 2006 was higher than that in 2002 (31.6%, 6/19 vs. 7.9%, 16/230). Areas near the boundary of Kaohsiung City and Fungshan City were the most severe epidemic regions, similar to the 2002 severe epidemic sites in Taiwan. The dynamic diffusion of dengue cases over the 2006 epidemic periods showed relocation besides the neighborhood spreading.
In the immuno-epidemiological investigation, we collected serum samples of 47 DF, 14 DHF and 14 healthy controls after the re-confirmation of their clinical category as DF and DHF cases by two experienced physicians of infections [mean±sd (range) of age: 47.6±17.4(5~74)、45.2±23.1(1~79) and 40.4±26.8 (12-79) years]. Symptoms/signs of classical dengue were found in more than 50% of dengue cases but DHF cases manifested more petechiae, loss of appetite and easily getting tired. Both DF and DHF patients had atypical lymphocytes and abnormal liver function (GOT: 71.53±47.75 vs. 38.67±17.43 units) only at defervescence stage, thrombocytopenia (112.20±89.36 vs. 126.69±86.34 K/ml). DHF compared to DF patients had prolonged activated partial thromboplastin time (APTT: 37.91±9.50 vs. 54.32±17.90 sec), more lymphocytes at acute and defervescence stage but lower numbers of monocytes at acute stage (9.00±4.46 vs. 5.23±2.53, p=0.022). About half (29/61, 47.5%) of dengue patients had chronic illness, particularly hypertension (14.8%, 9/61) and diabetes (11.5%, 7/61).
Virological and immunological analysis found that secondary dengue virus infection was more common than primary infection (77.0%, 47/61) in all dengue patients. Higher viral load was found in acute phase (n=18) than defervescence (n=25) of those dengue patients with secondary infection patients [RNA mean±SD: 3.16±2.57 log copies/ml vs.0.76±1.55 log copies/ml, p=0.001), and the similar results were found in the all DF patients (p=0.001). Additionally, the levels of the 5 cytokines (IL-1β, IL-2, IL-4, IL-10, TNF-α, IFN-γ) and 4 chemokines (RANTES, IP-10, MCP-1, MIG) of DF patients were higher than those of DHF patients whereas the levels of the IL-6 and IL-8 were much higher in DHF patients (IL-6: 6.5±5.1 vs. 20.7±16.2 pg/ml; IL-8: 85.8±306.1 vs. 498.2±1179.0 pg/ml). Additionally, the levels of IL-2 and IFN-γ in defervescence stage of DF patients were higher than those of DHF patients (IL-2: 8.6±6.2 pg/ml vs. 4.1±4.9 pg/ml, p=0.078; 52.6±141.8 vs. 8.8±8.1pg/ml, p=0.038). It indicates that immune activation of T cells decreased once the clinical severity increased. Particularly, IL-2 levels at 1-3 days after onset of illness interacted with viral load and RANTES in acute phase were negatively related to the duration of illness [β slope=0.09 (p=0.02) and β=-0.04 (p=0.03) in multiple linear regression after controlling of age, co-morbidity and days of rash. Furthermore, viral load was positively associated with the duration of illness after controlling of age, co-morbidity and days of rash. In other words, any of the three conditions including: (1) decreasing of IL-2 and RNATES without changes of viral load, or (2) decreasing IL-2 concomitant with increasing of viral load without changes of RANTES, or (3) increasing viral load that would lead to long duration of illness, serving for prognosis of clinical severity. Interestingly, Moreover, the level of IL-2 elevated at later period of the epidemic (>37 wk) (β=4.52, p=0.03) but declined at areas with increasing of transmission intensity (β=-4.94, p=0.02) (R2adj=0.22). The levels of inflammatory cytokine TNF-α in acute phase also negatively correlated with the interaction between duration of an epidemic wave and intensity of transmission (β=-2.31, p=0.03) (R2adj =0.15). Therefore, we suggested that population-based dynamic changes of immune responses rather than viral load occurred at different periods and various geographical areas during the epidemic of dengue/DHF.
In conclusion, defects in the levels of IL-2 and IFN-γ found at defervescence of DHF patients and the activation of IL-6 and IL-8 in Taiwan were different from the “cytokine cascade only” reported in the sera of severe DHF/DSS patients in South East Asian countries where dengue was endemic or hyper-endemic. This implies that different environments of epidemic areas might involve different mechanisms of immuno-pathogenesis of DHF. Future efforts are needed to measure the levels of innate immunity and type I interferon at different time points of infectious process to fully understand the causal inference of series interactions between viral and host factors under different environmental conditions that enhance the immunopathogenesis of DHF.

並列關鍵字

dengue fever/dengue hemorrhagic fever ； viral load and disease progression ； mechanism of immuno-pathogenesis ； cytokine/chemokine ； mechanism of epidemiological severity ； vector-born infectious disease ； Taiwan

參考文獻

Hwang, K.P., Su, S.C. and Chiang, C.H. (1989) [Clinical observations of dengue fever among children]. Gaoxiong Yi Xue Ke Xue Za Zhi 5, 50-7.

Ko, Y.C. (1989) [Epidemiology of dengue fever in Taiwan]. Gaoxiong Yi Xue Ke Xue Za Zhi 5, 1-11.

林鶯熹, 吳淑靜, 徐爾烈, 鄧華真, 何兆美, 白秀華 2002年台灣登革熱流行區埃及斑蚊的抗藥性. 台灣昆蟲. 2003;23:11.

Azeredo, E.L., Zagne, S.M., Santiago, M.A., Gouvea, A.S., Santana, A.A., Neves-Souza, P.C., Nogueira, R.M., Miagostovich, M.P. and Kubelka, C.F. (2001) Characterisation of lymphocyte response and cytokine patterns in patients with dengue fever. Immunobiology 204, 494-507.

Bethell, D.B., Flobbe, K., Cao, X.T., Day, N.P., Pham, T.P., Buurman, W.A., Cardosa, M.J., White, N.J. and Kwiatkowski, D. (1998) Pathophysiologic and prognostic role of cytokines in dengue hemorrhagic fever. J Infect Dis 177, 778-82.