Division of Infectious Diseases, Department of Medicine, University of North Carolina, 130 Mason Farm Rd, Chapel Hill, NC, 27599, USA.
Department of Epidemiology, Gillings School of Global Public Health, University of North Carolina, 135 Dauer Dr, Chapel Hill, NC, 27599, USA.
Malar J. 2018 Apr 2;17(1):137. doi: 10.1186/s12936-018-2287-4.
Rapid diagnostic tests (RDTs) that detect histidine-rich protein 2 (PfHRP2) are used throughout Africa for the diagnosis of Plasmodium falciparum malaria. However, recent reports indicate that parasites lacking the pfhrp2 and/or histidine-rich protein 3 (pfhrp3) genes, which produce antigens detected by these RDTs, are common in select regions of South America, Asia, and Africa. Proving the absence of a gene is challenging, and multiple PCR assays targeting these genes have been described. A detailed characterization and comparison of published assays is needed to facilitate robust and streamlined testing approaches.
Among six pfhrp2 and pfhrp3 PCR assays tested, the lower limit of detection ranged from 0.01 pg/µL to 0.1 ng/µL of P. falciparum 3D7 strain DNA, or approximately 0.4-4000 parasite genomes/µL. By lowering the elongation temperature to 60 °C, a tenfold improvement in the limit of detection and/or darker bands for all exon 1 targets and for the first-round reaction of a single exon 2 target was achieved. Additionally, assays targeting exon 1 of either gene yielded spurious amplification of the paralogous gene. Using these data, an optimized testing algorithm for the detection of pfhrp2- and pfhrp3-negative P. falciparum is proposed.
Surveillance of pfhrp2- and pfhrp3-negative P. falciparum requires careful laboratory workflows. PCR-based testing methods coupled with microscopy and/or antigen testing serve as useful tools to support policy development. Standardized approaches to the detection of pfhrp2- and pfhrp3-negative P. falciparum should inform efforts to define the impact of these parasites.
快速诊断检测（RDT）可检测富含组氨酸蛋白 2（PfHRP2），在整个非洲被用于诊断恶性疟原虫疟疾。然而，最近的报告表明，在南美洲、亚洲和非洲的一些特定地区，缺乏产生这些 RDT 检测抗原的 pfhrp2 和/或富含组氨酸蛋白 3（pfhrp3）基因的寄生虫很常见。证明基因缺失具有挑战性，并且已经描述了多种针对这些基因的 PCR 检测方法。需要对已发表的检测方法进行详细的特征描述和比较，以促进稳健且简化的检测方法。
在所测试的六种 pfhrp2 和 pfhrp3 PCR 检测方法中，检测下限范围为 0.01 pg/µL 至 0.1 ng/µL 恶性疟原虫 3D7 株 DNA，或约 0.4-4000 个寄生虫基因组/µL。通过将延伸温度降低至 60°C，可以将所有外显子 1 靶标和单个外显子 2 靶标的第一轮反应的检测下限提高十倍，并且条带也更暗。此外，针对这两个基因的外显子 1 的检测方法会导致同源基因的非特异性扩增。利用这些数据，提出了一种用于检测 pfhrp2-和 pfhrp3-阴性恶性疟原虫的优化检测算法。
对 pfhrp2-和 pfhrp3-阴性恶性疟原虫的监测需要仔细的实验室工作流程。基于 PCR 的检测方法结合显微镜检查和/或抗原检测，是支持政策制定的有用工具。检测 pfhrp2-和 pfhrp3-阴性恶性疟原虫的标准化方法应有助于确定这些寄生虫的影响。
