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Molecular Epidemiology of Anopheles coluzzii and Plasmodium falciparum in the Democratic Republic of Sao Tome and Principe
Democratic Republic of Sao Tome and Principe,Malaria,Anopheles coluzzii,Plasmodium falciparum,Molecular epidemiology,Genetic diversity,Resistance genes,
|Publication Year :||2020|
在瘧原蟲調查方面，本研究選取中央醫院病人的乾燥濾紙血片，使用恆溫環形核酸增幅法 (LAMP) 及定量即時聚合酶鏈鎖反應 (Q-PCR)，共檢測107個低密度感染 (鏡檢陰性但快篩陽性) 血片。結果顯示在低密度感染樣本中，LAMP及Q-PCR共檢測到78.5% 陽性，Q-PCR的方法偵測極限約為2隻瘧原蟲/微升DNA。另一方面，以裂殖體表面蛋白第一型 (MSP1) 及第二型 (MSP2) 序列，分析聖國惡性瘧原蟲流行株隨時間的變化，並針對111個治療前、後的血片樣本，做抗藥性基因分析，包含多重抗藥性蛋白 (pfmdr1)、氯奎寧抗性轉運蛋白 (pfcrt)、及Kelch樣蛋白13 (pfK13)。結果顯示當地的瘧原蟲流行株在近期的疫情高峰 (2012年) 出現轉變，瘧原蟲的MSP1由K1轉變為MAD20基因型為主，MSP2則由3D7/IC轉變為FC27基因型為主。在2014至2016年的檢體中，與第一線青蒿素搭配用藥阿莫待奎 (amodiaquine) 之抗藥性有關的主要點突變，包含pfmdr1 86Y及pfcrt 76T，其盛行率分別為82.9%及92.8%，普遍現存於當地的瘧原蟲族群。此外，與二線搭配用藥－苯芴醇 (lumefantrine) 之抗藥性有關的次要基因型，包含pfmdr1 184F及pfmdr1 D1246，亦流行於當地瘧原蟲株，其盛行率分別為62.2%及96.4%。然而，沒有任何與青蒿素抗藥性相關之突變在pfK13標的基因被偵測到。
最後，聖國如欲維持瘧疾清除前期，除需符合世界衛生組織定義的發燒病人年玻片陽性率 < 5% 外，根據本研究的流行病學結果，尚須達到以下標準：(a) 年發生率 < 1% (年個案數 < 2000例)、(b) 年平均室外蚊子密度 (人體誘餌法) < 1.6 隻/房/小時/月、(c) 針對奎寧治療的住院孩童，加強治療後的追蹤。本研究建議使用無抗藥性的病媒綜合管制法加強控制戶外帶有抗性的蚊子。分子檢驗方法可做為瘧疾清除前期下，檢測低密度感染的輔助工具。雖然聖國目前的瘧疾治療成效尚可接受，但研究結果發現當地瘧原蟲流行株的流變，並發展出抗藥性基因型，需加強治療成效的監測，以適時調整未來治療策略。
The Democratic Republic of Sao Tome and Principe (DRSTP) is an African island nation moving toward malaria pre-elimination phase. This study used molecular approach to survey the local vector Anopheles mosquitoes, malaria parasite Plasmodium falciparum, and their implications in malaria epidemiology. Genomic DNA of malaria vector was individually extracted from 1,923 female mosquitoes collected from 2010 to 2016. Parasite DNA was extracted from 218 dried blood spots (DBS) collected from malaria cases during 2014 to 2016. In the survey of mosquitoes, vector density was calculated by human landing catches and mosquito light traps. Genetic diversity and insecticide resistance of mosquitoes were analyzed by cytochrome c oxidase subunit I (COI), and knockdown resistance (kdr) mutation, respectively. Results showed that the malaria vector in the DRSTP is the newly named species, An. coluzzii with exophilic feature (previously named An. gambiae M form). Significant vector population differentiation was found between Principe and Sao Tome islands. The COI diversity and kdr mutation frequency of An. coluzzii were relatively low in Principe (mean of kdr freq. = 15.8%, n = 156) compared to Sao Tome (mean of kdr freq. = 44.8%, n = 1,767). The outdoor mosquito density and kdr mutation frequency increased to the highest during 2012 to 2013, which was corresponded with the malaria epidemic peak at the same time.
In the survey of parasites, DBS samples were selected from malaria patients administered to the Central Hospital. A total of 107 samples with low density infections (negative in microscopy but positive in rapid diagnostic test) were tested by loop-mediated isothermal amplification (LAMP) and quantitative PCR (Q-PCR). Results showed that LAMP and Q-PCR detected 78.5% of positive infections in the low density samples. The detection limit for Q-PCR assay was estimated to be ~2 parasites/μL DNA. On the other hand, temporal change of circulated parasite strains was identified by sequence analysis of merozoite surface protein 1 and 2 (MSP1 and MSP2). Anti-malarial drug resistance markers were also monitored including multi-drug resistance (pfmdr1), chloroquine resistance transporter (pfcrt), and kelch 13 (pfK13) in a total of 111 pre- and post-treated samples. The circulated parasite strains in the DRSTP showed significant temporal change at the epidemic peak in 2012, which the prevalent type in MSP1 changed from K1 to MAD20, and MSP2 changed from 3D7/IC to FC27 allelic type. Genotyping results of drug resistance markers showed the primary mutations associated with reduced sensitivity in the first-line partner drug, amodiaquine, were constantly presented in the local parasite population, with prevalence of 82.9% in pfmdr1 86Y; and 92.8% in pfcrt 76T. The secondary markers associated with the resistance of lumefantrine (second-line partner drug), were also found to be prevalent in the local parasites, with prevalence of 62.2% in pfmdr1 184F; and 96.4% in pfmdr1 D1246. No mutations were found in the artemisinin resistance marker, pfK13.
To pre-eliminate malaria, the country should achieve not only the criterion of slide positivity rate < 5% in fever cases defined by World Health Organization, but also sustain the following criteria based on our epidemiological results: (a) annual incidence rate < 1% (case numbers < 2000), (b) annual average outdoor density of human landing catches < 1.6 mosquitoes/house/hr/month, (c) strengthen the follow-ups of quinine-treated hospitalized children. This study suggests the control of outdoor resistant mosquitoes should be reinforced by resistance-free integrated vector management. Molecular diagnostic methods could become supplementary tools to detect low-density infections under pre-elimination phase. Although treatment efficacy remained acceptable in the DRSTP, this study found local parasites underwent temporal changes of dominant strains and development of drug resistance mutations. Therapeutic efficacy should be carefully monitored to adequately adjust treatment policy in the future.
|Appears in Collections:||環境與職業健康科學研究所|
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