Vallejo Andrés F, Rubiano Kelly, Amado Andres, Krystosik Amy R, Herrera Sócrates, Arévalo-Herrera Myriam
Malaria Vaccine and Drug Development Center (MVDC), Cali, Valle de Cauca, Colombia.
Caucaseco Scientific Research Center, Cali, Cali, Valle de Cauca, Colombia.
PLoS Negl Trop Dis. 2016 Jun 29;10(6):e0004807. doi: 10.1371/journal.pntd.0004807. eCollection 2016 Jun.
Individuals exposed to malaria infections for a long time develop immune responses capable of blocking Plasmodium transmission to mosquito vectors, potentially limiting parasite spreading in nature. Development of a malaria TB vaccine requires a better understanding of the mechanisms and main effectors responsible for transmission blocking (TB) responses. The lack of an in vitro culture system for Plasmodium vivax has been an important drawback for development of a standardized method to assess TB responses to this parasite. This study evaluated host, vector, and parasite factors that may influence Anopheles mosquito infection in order to develop an efficient and reliable assay to assess the TB immunity.
METHODS/PRINCIPAL FINDINGS: A total of 94 P. vivax infected patients were enrolled as parasite donors or subjects of direct mosquito feeding in two malaria endemic regions of Colombia (Tierralta, and Buenaventura). Parasite infectiousness was assessed by membrane feeding assay or direct feeding assay using laboratory reared Anopheles mosquitoes. Infection was measured by qPCR and by microscopically examining mosquito midguts at day 7 for the presence of oocysts. Best infectivity was attained in four day old mosquitoes fed at a density of 100 mosquitos/cage. Membrane feeding assays produced statistically significant better infections than direct feeding assays in parasite donors; cytokine profiles showed increased IFN-γ, TNF and IL-1 levels in non-infectious individuals. Mosquito infections and parasite maturation were more reliably assessed by PCR compared to microscopy.
We evaluated mosquito, parasite and host factors that may affect the outcome of parasite transmission as measured by artificial membrane feeding assays. Results have led us to conclude that: 1) optimal mosquito infectivity occurs with mosquitoes four days after emergence at a cage density of 100; 2) mosquito infectivity is best quantified by PCR as it may be underestimated by microscopy; 3) host cellular immune response did not appear to significantly affect mosquito infectivity; and 4) no statistically significant difference was observed in transmission between mosquitoes directly feeding on humans and artificial membrane feeding assays.
长期接触疟疾感染的个体产生的免疫反应能够阻断疟原虫向蚊媒的传播，这可能会限制寄生虫在自然界中的传播。开发疟疾传播阻断疫苗需要更好地了解负责传播阻断（TB）反应的机制和主要效应因子。缺乏间日疟原虫的体外培养系统一直是开发评估针对该寄生虫的TB反应的标准化方法的一个重要障碍。本研究评估了可能影响按蚊感染的宿主、蚊媒和寄生虫因素，以开发一种高效可靠的检测方法来评估TB免疫力。
方法/主要发现：在哥伦比亚的两个疟疾流行地区（蒂拉尔塔和布埃纳文图拉），共招募了94名间日疟原虫感染患者作为寄生虫供体或直接用于蚊虫叮咬的对象。通过膜饲法或使用实验室饲养的按蚊进行直接饲法评估寄生虫的传染性。在第7天通过qPCR和显微镜检查蚊中肠是否存在卵囊来测量感染情况。用4日龄的蚊子，以100只/笼的密度进行饲育，可获得最佳感染率。在寄生虫供体中，膜饲法产生的感染在统计学上显著优于直接饲法；细胞因子谱显示非感染个体中IFN-γ、TNF和IL-1水平升高。与显微镜检查相比，通过PCR能更可靠地评估蚊虫感染和寄生虫成熟情况。
我们评估了可能影响通过人工膜饲法测量的寄生虫传播结果的蚊虫、寄生虫和宿主因素。结果使我们得出以下结论：1）羽化后4天、笼密度为100只时的蚊子具有最佳感染性；2）PCR是量化蚊虫感染性的最佳方法，因为显微镜检查可能会低估感染性；3）宿主细胞免疫反应似乎并未显著影响蚊虫感染性；4）直接以人类为食的蚊子与人工膜饲法之间在传播方面未观察到统计学上的显著差异。
