Infectious Diseases Programme, Queensland Institute of Medical Research, Herston, Queensland, Australia ; School of Medicine, University of Queensland, St Lucia, Queensland, Australia.
PLoS One. 2013 Oct 2;8(10):e77811. doi: 10.1371/journal.pone.0077811. eCollection 2013.
The pre-erythrocytic stages of Plasmodium spp. are increasingly recognised as ideal targets for prophylactic vaccines and drug treatments. Intense research efforts in the last decade have been focused on in vitro culture and in vivo detection and quantification of liver stage parasites to assess the effects of candidate vaccines or drugs. Typically, the onset of blood stage parasitaemia is used as a surrogate endpoint to estimate the efficacy of vaccines and drugs targeting pre-erythrocytic parasite stages in animal models. However, this provides no information on the parasite burden in the liver after vaccination or treatment and therefore does not detect partial efficacy of any vaccine or drug candidates. Herein, we describe a quantitative RT-PCR method adapted to detect and quantitate Plasmodium yoelii liver stages in mice with increased sensitivity even after challenge with as few as 50 cryopreserved sporozoites (corresponding to approximately 5-10 freshly isolated sporozoites). We have validated our quantitative RT-PCR assay according to the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines and established high reproducibility and accuracy. Our assay provides a rapid and reproducible assessment of liver stage parasite burden in rodent malaria models, thereby facilitating the evaluation of the efficacy of anti-malarial drugs or prophylactic vaccines with high precision and efficacy.
疟原虫的红细胞前期阶段越来越被认为是预防疫苗和药物治疗的理想靶点。在过去十年中，人们进行了大量的研究工作，致力于体外培养和体内检测和定量肝期寄生虫，以评估候选疫苗或药物的效果。通常，血液期寄生虫血症的发生被用作替代终点来估计针对红细胞前期寄生虫阶段的疫苗和药物的疗效在动物模型中。然而，这不能提供疫苗接种或治疗后肝脏中寄生虫负荷的信息，因此无法检测任何疫苗或药物候选物的部分疗效。本文中，我们描述了一种定量 RT-PCR 方法，该方法经过改良，即使在仅用 50 个冷冻保存的孢子（相当于大约 5-10 个新鲜分离的孢子）进行挑战后，也能提高检测和定量小鼠中的疟原虫 yoelii 肝期的灵敏度。我们根据 MIQE（定量实时 PCR 实验的最低信息要求）指南验证了我们的定量 RT-PCR 检测方法，并建立了高重复性和准确性。我们的检测方法为啮齿动物疟疾模型中的肝期寄生虫负荷提供了快速和可重复的评估，从而能够精确和高效地评估抗疟药物或预防性疫苗的疗效。
