Division of Medical Microbiology, Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
J Clin Microbiol. 2013 Sep;51(9):2931-8. doi: 10.1128/JCM.00958-13. Epub 2013 Jun 26.
Molecular diagnosis of malaria offers many potential advantages over microscopy, including identification of malaria to the species level in an era with few experienced microscopists. We developed high-throughput multiplex 5' nuclease quantitative PCR (qPCR) assays, with the potential to support large studies, to specifically identify Plasmodium falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. We compared qPCR to microscopy and confirmed discordant results with an alternative target PCR assay. The assays specifically detected 1 to 6 parasites/μl of blood. The clinical sensitivities (95% confidence intervals [CIs]) of the 4-plex assay to detect microscopically confirmed malaria were 95.8% (88.3 to 99.1%) for P. falciparum, 89.5% (75.2 to 97.1%) for P. vivax, 94.1% (71.3 to 99.9%) for P. ovale, and 100% (66.4 to 100%) for P. malariae. The specificities (95% CIs) were 98.6% (92.4 to 100%) for P. falciparum, 99% (84.8 to 100%) for P. vivax, 98.4% (94.4 to 99.8%) for P. ovale, and 99.3% (95.9 to 100%) for P. malariae. The clinical specificity for samples without malaria was 100%. The clinical sensitivity of the 5-plex assay for confirmed P. knowlesi malaria was 100% (95% CI, 69.2 to 100%), and the clinical specificity was 100% (95% CI, 87.2 to 100%). Coded retesting and testing with an alternative target PCR assay showed improved sensitivity and specificity of multiplex qPCR versus microscopy. Additionally, 91.7% (11/12) of the samples with uncertain species by microscopy were identified to the species level identically by both our multiplex qPCR assay and the alternative target PCR assay, including 9 P. falciparum infections. Multiplex qPCR can rapidly and simultaneously identify all 5 Plasmodium species known to cause malaria in humans, and it offers an alternative or adjunct to microscopy for clinical diagnosis as well as a needed high-throughput tool for research.
疟疾的分子诊断与显微镜相比具有许多潜在的优势，包括在经验丰富的显微镜检师较少的时代能够鉴定到物种水平。我们开发了高通量多重 5' 核酸酶定量 PCR（qPCR）检测法，具有支持大规模研究的潜力，可特异性鉴定恶性疟原虫、间日疟原虫、卵形疟原虫、三日疟原虫和猴疟原虫。我们将 qPCR 与显微镜检查进行了比较，并通过另一种靶标 PCR 检测法确认了不一致的结果。该检测法可特异性检测到每微升血液中 1 至 6 个寄生虫。4 重检测法检测经显微镜确认的疟疾的临床灵敏度（95%置信区间 [CI]）分别为恶性疟原虫 95.8%（88.3%至 99.1%）、间日疟原虫 89.5%（75.2%至 97.1%）、卵形疟原虫 94.1%（71.3%至 99.9%）和三日疟原虫 100%（66.4%至 100%）。特异性（95%CI）分别为恶性疟原虫 98.6%（92.4%至 100%）、间日疟原虫 99%（84.8%至 100%）、卵形疟原虫 98.4%（94.4%至 99.8%）和三日疟原虫 99.3%（95.9%至 100%）。无疟疾样本的临床特异性为 100%。经确认的伯氏疟原虫感染的 5 重检测法的临床灵敏度为 100%（95%CI，69.2%至 100%），临床特异性为 100%（95%CI，87.2%至 100%）。经编码重新检测和使用替代靶标 PCR 检测显示，与显微镜相比，多重 qPCR 的灵敏度和特异性均得到改善。此外，12 份经显微镜检查不确定物种的样本中有 9 份（91.7%）通过我们的多重 qPCR 检测法和替代靶标 PCR 检测法同样鉴定到物种水平，其中包括 9 份恶性疟原虫感染。多重 qPCR 可以快速、同时鉴定出所有已知的 5 种引起人类疟疾的疟原虫，它为临床诊断提供了显微镜检查的替代方法或辅助方法，也是研究所需的高通量工具。
