聖多美普林西比民主共和國斑點熱立克次體之分子及血清流行病學研究

Abstract

斑點熱立克次體病是一種新興以及再興蟲媒傳染病，病原體屬於立克次體屬，主要以硬蜱作為主要的病媒。聖多美普林西比民主共和國是中非熱帶地區島國，當地居民的居住環境靠近農場與叢林，與家畜及其寄生性節肢動物接觸頻率高，形成有利於蜱傳播斑點熱的生活環境。在過去的斑點熱血清流行病研究中，撒哈拉沙漠以南非洲地區的盛行率從0% 到91.4%不等，但是在聖多美並沒有斑點熱的相關報告。除此之外，聖多美近年的瘧疾檢驗陽性率已經大幅下降，但是去醫院就診的不明原因發燒病人數卻年年上升。這些非感染瘧疾的發燒病人有否可能是由於潛在的斑點熱立克次體感染所造成，值得進一步研究。在先期試驗中，以124個長期居住在聖多美的婦女血清為樣本，透過酵素免疫分析法檢測IgG抗體，血清陽性率為8.9%。在進一步的橫斷式研究中，於2016年在省區Agua Grande收集的健康居民乾燥濾紙血片檢體以不同年齡層進行隨機抽樣選取240個檢體，以間接免疫螢光染色法檢測抗斑點熱立克次體IgG抗體。結果顯示對病原體R. africae, R. conorii, R. felis 的血清陽性率分別為1.7% (4/240)，6.7% (16/240)，以及 2.9% (7/240)。在八個不同年齡層中，0-5歲的血清陽性率為13.3% (4/30)，16-25歲為 20.0% (6/30)，26-35歲為16.7% (5/30)，46-55歲為6.7% (2/30)，56-65歲為13.3% (4/30)，年齡>65的族群陽性率為23.3%(7/30)，其中6-15歲及36-45歲沒有檢測到陽性檢體。斑點熱立克次體的總血清陽性率為11.7% (28/240)，其中>65歲的年齡層有最高的陽性率。外寄生蜱分別於2012年在聖多美南部的一座動物牧場，以及2016年在聖多美北部Lobata省區的Agua Casada動物牧場從牛羊身上直接採集。102隻硬蜱檢體以形態學鑑定，2016年採集的60個檢體為變異花蜱(Amblyomma variegatum)，2012年採集的42個檢體為星狀花蜱(Amblyomma astrion)。蜱體內的立克次體以聚合酶連鎖反應(PCR)檢測立克次體的gltA，ompA，ompB基因，變異花蜱的陽性率為81.7% (49/60)，星狀花蜱陽性率為 52.4% (22/42)。蜱體內立克次體的種類鑑定是依據Fournier and other於2003年提出的指引，定序立克次體的rrs，gltA，ompA，ompB，以及sca4基因，並進行序列比對。變異花蜱檢體中定序的五段立克次體基因序列與R. africae ESF-5有最高的相似度。各基因序列的相似度為rrs 基因 99.1%，gltA 基因 98.7% ， 5’ 端ompA基因98.6%，ompB基因99.1%，以及sca4基因99.0%。上述基因序列的相似度均低於同種的判定數值，初步鑑定為新種立克次體。藉由血清流行病學以及分子學資料顯示，目前斑點熱立克次體在聖多美盛行，本研究結果將提供聖多美斑點熱立克次體感染的證據，提供公衛部門及醫師診治之參考。

Spotted fever group (SFG) rickettsioses are emerging and reemerging vector-borne disease around the world. Rickettsioses are caused by bacteria belonging to the genus Rickettsia, and are mainly transmitted by Ixodid ticks. In the tropic African country, Democratic Republic of Sao Tome and Principe (DRSTP), most residents live beside the jungles and farms and therefore contact frequently with animals and insects, which implicate a suitable environment for tick populations. The sero-prevalence of SFG rickettsioses in sub-Sahara Africa was about 0% to 91.4% in previous seroepidemiological studies. However, there are no reports in the DRSTP. Furthermore, the incidence of malaria there is decreasing significantly in recent years, but the number of patients with fever of unknown origin (FUO) was still high. Thus, there could be a high possibility of SFG rickettsiae infection that happens on local residents with FUO. Preliminary test was performed by ELISA against SFG IgG, and sero-positive rate was 8.9% in 124 serum samples from pregnant women. In further cross-sectional study, dried blood spots (DBS) samples from healthy people were collected from January 2016 to March 2016 in Agua Grande. The indirect immunofluorescence assay (IFA) was used to detect IgG antibodies against SFG rickettsiae in 240 DBS samples randomly chosen by age groups. The sero-positive rate was 1.7% (4/240), 6.7% (16/240), and 2.9% (7/240) in R. africae, R. conorii, and R. felis, respectively. And the total SFG sero-positive rate was 11.7% (28/240). In the 8 age groups, the sero-positive rate were 13.3%(4/30) in 0-5, 0% in 6-15, 20.0%(6/30) in 16-25, 16.7%(5/30) in 26-35, 0% in 36-45, 6.7%(2/30) in 46-55, 13.3%(4/30) in 56-65, and 23.3%(7/30) in age >65. The highest sero-positive rate was in age group >65. The highest sero-positive rate was found in older people. Ectoparasites were collected from cattles and goats directly in the Agua Casada animal farm located in the province of Lobata in northern sao tome in 2016 and in a farm located in southern sao tome in 2012. A total number of 102 ixodid ticks were identified based on morphological characteristics, 42 ticks collected in 2012 were morphologically identified as Amblyomma astrion, and 60 ticks collected in 2016 were identified as Amblyomma variegatum. The infection rate of rickettsiae in ticks was detected by using polymerase chain reaction targeting the gltA, ompA and ompB genes. The infection rate was 81.7% (49/60) and 52.4% (22/42) in A. variegatum and A. astrion, respectively. The Rickettsia species identification followed the guidelines proposed by Fournier and other (2003), sequences of rrs, gltA, ompA, ompB, and sca4 gene were sequenced and BLAST. The validated Rickettsia species R.africae ESF-5 (CP001612) was the most similar species among the identity of the five rickettsial gene sequences from A. variegatum. The identities with R. africae ESF-5 (CP001612.1) were 99.06% for rrs gene, 98.7% for gltA gene, 98.6% for 5’ end ompA gene, 99.1% for ompB gene, and 99.0% for sca4 gene. All the identities are not higher than the cut-off value for identification. Based on the criteria, the sequences obtained in this study should belong to a novel Rickettsia species. The serological and molecular evidence in this study supports that SFG rickettsiae were prevalent in the DRSTP. The evidence could provide the public health department and clinics for the risk of SFG exposure.