Swiss Tropical and Public Health Institute, Socinstrasse 57, 4051, Basel, Switzerland.
University of Basel, Basel, Switzerland.
Malar J. 2020 Jan 29;19(1):50. doi: 10.1186/s12936-020-3127-x.
Molecular detection of low-density Plasmodium falciparum infections is essential for surveillance studies conducted to inform malaria control strategies in close-to-elimination settings. Molecular monitoring of residual malaria infections usually requires a large study size, therefore sampling and diagnostic processes need to be economical and optimized for high-throughput. A method comparison was undertaken to identify the most efficient diagnostic procedure for processing large collections of community samples with optimal test sensitivity, simplicity, and minimal costs.
In a reactive case detection study conducted on Zanzibar, parasitaemia of 4590 individuals of all ages was investigated by a highly sensitive quantitative (q) PCR that targets multiple var gene copies per parasite genome. To reduce cost, a first round of positivity screening was performed on pools of dried blood spots from five individuals. Ten cycles of a pre-PCR were performed directly on the filter paper punches, followed by qPCR. In a second round, samples of positive pools were individually analysed by pre-PCR and qPCR.
Prevalence in household members and neighbors of index cases was 1.7% (78/4590) with a geometric mean parasite density of 58 parasites/µl blood. Using qPCR as gold standard, diagnostic sensitivity of rapid diagnostic tests (RDTs) was 37% (29/78). Infections positive by qPCR but negative by RDT had mean densities of 15 parasites/µl blood.
The approach of pre-screening reactive case detection samples in pools of five was ideal for a low prevalence setting such as in Zanzibar. Performing direct PCR on filter paper punches saves substantial time and justifies the higher cost for a polymerase suitable for amplifying DNA directly from whole blood. Molecular monitoring in community samples provided a more accurate picture of infection prevalence, as it identified a potential reservoir of infection that was largely missed by RDT. The developed qPCR-based methodology for screening large sample sets represents primarily a research tool that should inform the design of malaria elimination strategies. It may also prove beneficial for diagnostic tasks in surveillance-response activities.
在接近消除疟疾的环境中，为了为疟疾控制策略提供信息，进行监测研究，检测低密度恶性疟原虫感染的分子检测至关重要。残留疟疾感染的分子监测通常需要较大的研究规模，因此采样和诊断过程需要经济实惠，并针对高通量进行优化。进行了方法比较，以确定用于处理具有最佳测试灵敏度、简单性和最小成本的大量社区样本的最有效诊断程序。
在桑给巴尔进行的一项反应性病例检测研究中，对所有年龄段的 4590 个人进行了高度敏感的定量（q）PCR 检测，该检测针对每个寄生虫基因组中的多个 VAR 基因拷贝。为了降低成本，对来自五个人的干燥血斑的阳性池进行了第一轮阳性筛选。在预 PCR 中进行了十个循环，直接在滤纸上打孔，然后进行 qPCR。在第二轮中，对阳性池的样本进行单独的预 PCR 和 qPCR 分析。
指数病例的家庭成员和邻居中的患病率为 1.7%（78/4590），几何平均寄生虫密度为 58 个寄生虫/µl 血液。使用 qPCR 作为金标准，快速诊断测试（RDT）的诊断灵敏度为 37%（29/78）。qPCR 阳性但 RDT 阴性的感染的平均密度为 15 个寄生虫/µl 血液。
在像桑给巴尔这样低流行率的环境中，对五个反应性病例检测样本的阳性池进行预筛选的方法是理想的。在滤纸上打孔上直接进行 PCR 可以节省大量时间，并证明适合直接从全血中扩增 DNA 的聚合酶的更高成本是合理的。社区样本中的分子监测提供了更准确的感染流行率图景，因为它识别出了 RDT 大大遗漏的感染潜在储存库。用于筛选大样本集的基于 qPCR 的方法主要是一种研究工具，它应该为疟疾消除策略的设计提供信息。它也可能对监测反应活动中的诊断任务有益。
