Lamien-Meda Aline, Fuehrer Hans-Peter, Noedl Harald
Institute of Specific Prophylaxis and Tropical Medicine, Medical University of Vienna, Austria.
Institute of Parasitology, University of Veterinary Medicine, Vienna, Austria.
Acta Trop. 2019 Apr;192:75-81. doi: 10.1016/j.actatropica.2019.01.018. Epub 2019 Jan 31.
Plasmodium ovale spp. are two of the six species of apicomplexan parasites belonging to the genus Plasmodium commonly causing disease in humans. A recent phylogeny study has identified both Plasmodium ovale species (P. ovale curtisi and P. ovale wallikeri) as two sympatric occurring species. The actual prevalence and clinical relevance of P. ovale spp. are likely underestimated due to low parasitemia and mixed infections, which pose a major challenge to microscopic diagnosis and are frequently undetectable using malaria Rapid Diagnostic Tests (RDTs). The aim of this work is to develop a HRM-based assay for simultaneous detection and differentiation of P. ovale wallikeri and P. ovale curtisi. Thirty three well-documented P. ovale spp. samples from previous studies were used for this study. The newly developed High Resolution Melting (HRM) assay targeting the apicoplast genome was highly specific to both P. ovale species. Adding a snapback tail at the 5' end of the forward primer for a nested HRM PCR, increased the melting temperature (Tm) difference between the two species. To our knowledge this study reports the first direct HRM assay developed on the apicoplast genome, specific for both P. ovale species. This method provides added value to the WHO open request of developing new practical malaria diagnostic methods for the malaria elimination program and could contribute to a quick and efficient diagnosis of low-level parasitemia, symptomatic or asymptomatic, as well as mixed or single P. ovale infections.
卵形疟原虫属是疟原虫属六种顶复门寄生虫中的两种，这些寄生虫通常会导致人类发病。最近的系统发育研究已将两种卵形疟原虫（柯氏疟原虫和沃氏疟原虫）确定为两个同域分布的物种。由于低虫血症和混合感染，卵形疟原虫属的实际流行率和临床相关性可能被低估，这给显微镜诊断带来了重大挑战，并且使用疟疾快速诊断检测（RDT）常常无法检测到。这项工作的目的是开发一种基于高分辨率熔解曲线分析（HRM）的检测方法，用于同时检测和区分沃氏疟原虫和柯氏疟原虫。本研究使用了先前研究中33个记录良好的卵形疟原虫属样本。新开发的针对质体基因组的高分辨率熔解曲线分析（HRM）检测方法对两种卵形疟原虫都具有高度特异性。在巢式HRM PCR的正向引物5'端添加一个回文尾，增加了两种之间的熔解温度（Tm）差异。据我们所知，本研究报告了首次在质体基因组上开发的直接HRM检测方法，对两种卵形疟原虫均具有特异性。该方法为世界卫生组织关于为疟疾消除计划开发新的实用疟疾诊断方法的公开要求提供了附加价值，并且有助于快速、高效地诊断低水平虫血症，无论是有症状还是无症状的，以及混合或单一的卵形疟原虫感染。
