- 學位論文
聖多美普林西比之新興或再興蟲媒傳染病流行病學研究
Epidemiological studies of emerging and re-emerging vector-borne infectious diseases in the Democratic Republic of Sao Tome and Principe
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摘要
聖多美普林西比民主共和國位於非洲西部幾內亞灣，過往飽受瘧疾侵擾，在臺灣瘧疾防治顧問團和聖國衛生部門合作下，自2003年起積極推動各項防治措施，瘧疾盛行率因而迅速下降，2013年的資料顯示，蘭巴(Lemba)行政區和普林西比自治區的血片檢查陽性率已低於5%，達到世界衛生組織訂定瘧疾清除前期的標準。然而，聖國當地因不明原因發燒至醫院就診的病人數卻未見減少。聖多美普林西比屬於熱帶雨林氣候，適合病媒節肢動物繁殖，人民的居住環境靠近農場與叢林，與蚊子或外寄生蟲接觸頻率高，但除了瘧疾外，其他蟲媒傳染病卻鮮少受到關注，本研究旨在探討聖國境內是否有其他新興或再興蟲媒傳染病傳播，造成公共衛生的潛在威脅。論文內容包括兩部分，分別以蚊子媒介的登革病毒、屈公病毒，以及恙蟎媒介的恙蟲病作為研究目標，利用血清學的方法調查蚊媒病毒和蟎媒立克次體的感染。論文的第一部分，使用間接免疫螢光染色法(immunofluorescence assay, IFA)和酵素鍵結免疫吸附方法(enzyme-linked immune-sorbent assay, ELISA)進行實驗，發現在2003-2004年間收集自長期居住於聖多美的懷孕婦女血清檢體中(n=78)，有28個(35.9%)樣本測得了登革病毒抗體，其中一個樣本呈現免疫球蛋白M (IgM)陽性，顯示採檢當時有近期的病毒暴露，另外，6個檢體則帶有屈公病毒抗體。由於蟲媒傳染病的防治目前主要仍倚賴對病媒密度的控制，自2000年起臺灣瘧疾防治顧問團例行於聖國進行病媒蚊調查，2016年首度在島上發現白線斑蚊，本研究利用型態學及分子鑑定方式，確定白線斑蚊入侵，全國調查的結果更證實白線斑蚊已遍布各省分。鑒於聖國原生的埃及斑蚊屬於森林型亞種(Ae. aegypti formosus)，並不嗜食人血，白線斑蚊的入侵因此更具有公共衛生的重要性。近來，蚊蟲特異性唾液蛋白抗體被證實可以當成蚊蟲叮咬頻度的工具和指標，並藉以評估疾病傳播的風險。因此本研究完成表現埃及斑蚊和白線斑蚊的重組蚊唾液蛋白(rAAE_34-kDa和rAAL_34-kDa)與合成短片段的特異性唾腺蛋白胜肽，將來可應用於釐清白線斑蚊入侵聖多美普林西比民主共和國的時間和擴散過程，以及評估未來登革熱在聖國傳播的風險。論文的第二部分分析恙蟲病感染，於2016年在聖多美島人口最密集的大水(Agua Grande)行政區收集乾燥濾紙血片(dried blood spots, DBS)，以間接免疫螢光染色法檢測，發現 240個樣品中，14個(5.8%)呈現恙蟲病立克次體抗體陽性。為了解恙蟲病在聖國的傳播，本研究進一步檢測了570個於2003年收集的孕婦血清，IFA和ELISA分別測得20.4%及15.5%的陽性率，IgM陽性比率並高達13.5%及18.5%，顯示恙蟲病可能在當地存在超過十年，卻長期受到忽視。雖然我們的研究尚未能找出聖國恙蟲病立克次體的確切傳播途徑，但以臺灣恙蟲病為例，主要的媒介動物為地里恙蟎(Leptotrombidium deliense)與小板盾恙蟎(L. scutellare)，有鑑於目前資料庫中恙蟎基因體仍不完整，本論文以次世代定序方式從頭定序(de novo sequencing)上述二種恙蟎基因體，雙邊定序(paired-end sequencing)的結果，共取得~340百萬筆讀值(reads)，定序深度分別達到地里恙蟎約37.1倍、小板盾恙蟎約35和40.9倍。估計地里恙蟎和小板盾恙蟎的基因體大小為190.6 Mb和236 Mb，GC比率分別為33.7%和33%。地里恙蟎序列組合的N50為1,084 bp，小板盾恙蟎則為1,098和1,001 bp。運用AUGUSTUS軟體預測蛋白編碼區域發現，恙蟲的訊息RNA(mRNA)長度跟表現子(exon)的數量都比蚊子(以埃及斑蚊為例)短少。期望未來能藉由研究中對恙蟲基因組解序，找出恙蟲的唾液組成基因，進而了解恙蟲叮咬與恙蟲立克次體感染的風險。總結以上，本論文首度證明聖多美普林西比民主共和國境內，不止瘧原蟲，也可能有登革熱、屈公熱和恙蟲病等蟲媒傳染病傳播，可作為醫師診斷的參考和當地衛生機構進行病媒防治的依據，更為全球蟲媒傳染病的分布提供了新的資訊。
並列摘要
The Democratic Republic of Sao Tome and Principe (DRSTP) is located in the Gulf of Guinea in West Africa. Malaria used to be the prevailing disease on the islands, but its incidence has significantly decreased after multiple interventions by the cooperation of the Taiwan Anti-Malaria Advisory Team and the Ministry of Health of the DRSTP since 2003. However, numerous patients still visited the hospitals due to acute undifferentiated febrile illness. With a tropical rain forest climate, the islands provide natural habitats for various arthropods. Local residents frequently expose to mosquito or ectoparasite bites by living near farms and jungles. Yet vector-borne infectious diseases received little attention except malaria. The study aimed to discover the hidden burden of public health caused by vector-borne diseases in the DRSTP. In the first part of the dissertation, the transmission of mosquito-borne viral diseases such as dengue and chikungunya was investigated. Of 78 serum samples collected from pregnant women lived in the nation, antibodies against dengue virus were detected in 28 (35.9%) samples by using indirect immunofluorescence assay (IFA) and enzyme-linked immune-sorbent assay (ELISA), and one of them was immunoglobulin M (IgM) positive, showing recent virus exposure at the time of sampling. On the other hand, antibodies against chikungunya virus were found in 6 samples. As the control of vector-borne diseases largely relies on reducing the vector densities, the Anti-Malaria Advisory Team routinely monitored mosquito populations in the DRSTP since 2000. The introduction of Aedes albopictus was reported on the island for the first time in 2016. This study used morphological characteristics and molecular identification to confirm the invasion of Ae. albopictus. Further nationwide survey showed that the Asian tiger mosquito has spread throughout all the provinces. As the native Ae. aegypti formosus is less anthropophilic, the invasive Ae. albopictus is of public health significance. Recently, several researches have adopted the antibody response to vector salivary proteins as an epidemiological indicator of exposure to vector bites and used it for assessing the risk of disease transmission. This study, therefore, has expressed two orthologous recombinant proteins: rAAE_34-kDa and rAAL_34-kDa as well as synthetic genus-specific peptides which could be applied for future analysis to uncover the details of the invasion of Ae. albopictus. The second part of the dissertation focused on mite-borne rickettsiosis. Dried blood spots (DBS) were collected from healthy residents of Agua Grande District during the proactive mass screening of malaria conducted in 2016. Among 240 samples screened by IFA, 14 (5.8%) samples were positive for Orientia tsutsugamushi antibody. Additional 570 serum samples from pregnant women collected in 2003 were also examined, resulting in a positive rates of 20.4% and 15.5% by IFA and ELISA, respectively. IgM positive rates were as high as 13.5% and 18.5%, respectively. These results implied that Orientia sp. might have circulated on the islands for more than a decade without being noticed. Although our study has not yet identified the natural transmission cycle of Orientia sp. in the DRSTP, it is believed that humans contract scrub typhus through chigger bites. The main vectors of O. tsutsugamushi are Leptotrombidium deliense and L. scutellare in Taiwan. Since the genome sequences of trombiculid mites are still incomplete in the current database, de novo sequencing was performed for the above two species. Assembled genome sizes for trombiculid mites were 190.6 Mb and 236 Mb, respectively. GC contents were 33.7% for L. deliense and averaged 33% for L. scutellare. Using the software AUGUSTUS to predict protein coding regions, it was found that the mRNA length and the number of exons of the trombiculid mites were shorter and less than those of mosquitoes (for example, Ae. aegypti). The sequences will be used to identify salivary genes of chiggers and help to further evaluate the risk of chigger bites as well as Orientia infections. In conclusion, these findings have demonstrated possible transmission of dengue virus, chikungunya virus, and Orientia sp. in the nation, providing a reference for differential diagnosis and background knowledge for disease control. Moreover, it provides more insight on the global distribution of these vector-borne diseases.
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