Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
PLoS One. 2013 Jul 22;8(7):e69781. doi: 10.1371/journal.pone.0069781. Print 2013.
To be able to robustly propagate P. falciparum at optimal conditions in vitro is of fundamental importance for genotypic and phenotypic studies of both established and fresh clinical isolates. Cryo-preserved P. falciparum isolates from Ugandan children with severe or uncomplicated malaria were investigated for parasite phenotypes under different in vitro growth conditions or studied directly from the peripheral blood. The parasite cultures showed a minimal loss of parasite-mass and preserved percentage of multiple infected pRBCs to that in peripheral blood, maintained adhesive phenotypes and good outgrowth and multiplication rates when grown in suspension and supplemented with gas. In contrast, abnormal and greatly fluctuating levels of multiple infections were observed during static growth conditions and outgrowth and multiplication rates were inferior. Serum, as compared to Albumax, was found necessary for optimal presentation of PfEMP1 at the pRBC surface and/or for binding of serum proteins (immunoglobulins). Optimal in vitro growth conditions of P. falciparum therefore include orbital shaking (50 rev/min), human serum (10%) and a fixed gas composition (5% O2, 5% CO2, 90% N2). We subsequently established 100% of 76 frozen patient isolates and found rosetting with schizont pRBCs in every isolate (>26% schizont rosetting rate). Rosetting during schizogony was often followed by invasion of the bound RBC as seen by regular and time-lapse microscopy as well as transmission electron microscopy. The peripheral parasitemia, the level of rosetting and the rate of multiplication correlated positively to one another for individual isolates. Rosetting was also more frequent with trophozoite and schizont pRBCs of children with severe versus uncomplicated malaria (p<0.002; p<0.004). The associations suggest that rosetting enhances the ability of the parasite to multiply within the human host.
为了能够在最佳条件下在体外大量繁殖恶性疟原虫，对于建立和新鲜临床分离株的基因型和表型研究至关重要。对来自乌干达患有严重或非复杂性疟疾儿童的冷冻保存恶性疟原虫分离物进行了研究，以在不同的体外生长条件下研究寄生虫表型，或直接从外周血中进行研究。寄生虫培养物显示出最小的寄生虫质量损失和保持多个感染的 RBC 的百分比与外周血中的相同，在悬浮状态下生长并补充气体时保持粘附表型和良好的生长和增殖率。相比之下，在静态生长条件下观察到异常和极大波动的多重感染水平，并且生长和增殖率较差。与 Albumax 相比，血清被发现对于 PfEMP1 在 RBC 表面的最佳呈现和/或血清蛋白（免疫球蛋白）的结合是必需的。因此，恶性疟原虫的最佳体外生长条件包括轨道晃动（50 转/分钟）、人血清（10%）和固定的气体组成（5%O2、5%CO2、90%N2）。我们随后建立了 100%的 76 个冷冻患者分离物，并发现每个分离物中都有裂殖体 RBC 的玫瑰花结（>26%的裂殖体玫瑰花结率）。在裂殖体期间形成玫瑰花结后，经常通过常规和延时显微镜以及透射电子显微镜观察到结合的 RBC 的入侵。单个分离物的外周寄生虫血症、玫瑰花结水平和倍增率彼此之间呈正相关。与非复杂性疟疾相比，严重疟疾儿童的滋养体和裂殖体 RBC 的玫瑰花结更为频繁（p<0.002；p<0.004）。这些关联表明玫瑰花结增强了寄生虫在人体宿主中繁殖的能力。
