Cellulose filtration of blood from malaria patients for improving ex vivo growth of Plasmodium falciparum parasites.
Cellulose filtration of blood from malaria patients for improving ex vivo growth of Plasmodium falciparum parasites.
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DOI：
10.1186/s12936-017-1714-2
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发表时间：
2017-02-10
期刊：
影响因子：
3
通讯作者：
Wang CW
中科院分区：
文献类型：
--
作者：
Mkumbaye SI;Minja DT;Jespersen JS;Alifrangis M;Kavishe RA;Mwakalinga SB;Lusingu JP;Theander TG;Lavstsen T;Wang CW
从患者寄生虫分离株中建立恶性疟原虫体外培养系可以提供对药物敏感性的地理差异以及疟疾发病机制和免疫机制的更深入理解。血液的纤维素柱过滤是一种廉价、快速和有效的方法，用于去除宿主因子，如白细胞和血小板，显著改善血液样品中寄生虫DNA的纯化。在这项研究中，静脉血的纤维素柱过滤对从住院的坦桑尼亚儿童分离的恶性疟原虫寄生虫的初始体外生长的影响进行了测试。允许寄生虫在培养物中扩增而不进行传代培养，直至入院后5天或每天确定死亡寄生虫和寄生虫血症的出现。为了研究过滤是否对克隆性有影响，使用巢式PCR对提取的基因组DNA进行恶性疟原虫裂殖子表面蛋白2基因分型，并使用定量PCR对提取的RNA在入院和培养4天时研究var基因转录水平。纤维素过滤的寄生虫比未过滤的寄生虫更快地生长到更高的寄生虫血症，似乎是由于更高的发展比率的环阶段寄生虫进展到后期阶段。纤维素过滤对克隆性或var基因表达没有明显影响;然而，与入院时相比，仅培养4天后，在克隆数量和var基因转录水平方面观察到明显差异。 寄生虫血液的纤维素柱过滤是一种廉价、适用的方法，可用于改进恶性疟原虫田间分离株的培养，以进行基于离体的试验;然而，在评估培养寄生虫的表型和基因型时，通常假设代表体内感染，建议谨慎。本文的在线版本（doi：10.1186/s12936-017-1714-2）包含补充材料，可供授权用户使用。
Establishing in vitro Plasmodium falciparum culture lines from patient parasite isolates can offer deeper understanding of geographic variations of drug sensitivity and mechanisms of malaria pathogenesis and immunity. Cellulose column filtration of blood is an inexpensive, rapid and effective method for the removal of host factors, such as leucocytes and platelets, significantly improving the purification of parasite DNA in a blood sample. In this study, the effect of cellulose column filtration of venous blood on the initial in vitro growth of P. falciparum parasite isolates from Tanzanian children admitted to hospital was tested. The parasites were allowed to expand in culture without subcultivation until 5 days after admission or the appearance of dead parasites and parasitaemia was determined daily. To investigate whether the filtration had an effect on clonality, P. falciparum merozoite surface protein 2 genotyping was performed using nested PCR on extracted genomic DNA, and the var gene transcript levels were investigated, using quantitative PCR on extracted RNA, at admission and 4 days of culture. The cellulose-filtered parasites grew to higher parasitaemia faster than non-filtered parasites seemingly due to a higher development ratio of ring stage parasites progressing into the late stages. Cellulose filtration had no apparent effect on clonality or var gene expression; however, evident differences were observed after only 4 days of culture in both the number of clones and transcript levels of var genes compared to the time of admission. Cellulose column filtration of parasitized blood is a cheap, applicable method for improving cultivation of P. falciparum field isolates for ex vivo based assays; however, when assessing phenotype and genotype of cultured parasites, in general, assumed to represent the in vivo infection, caution is advised. The online version of this article (doi:10.1186/s12936-017-1714-2) contains supplementary material, which is available to authorized users.