开发用于检测和监测恶性疟原虫中与哌喹耐药相关的膜蛋白酶 2/3 拷贝数变异的拷贝数检测方法。
Development of copy number assays for detection and surveillance of piperaquine resistance associated plasmepsin 2/3 copy number variation in Plasmodium falciparum.
机构信息
Wellcome Sanger Institute, Hinxton, UK.
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, USA.
出版信息
Malar J. 2020 May 13;19(1):181. doi: 10.1186/s12936-020-03249-x.
BACKGROUND
Long regarded as an epicenter of drug-resistant malaria, Southeast Asia continues to provide new challenges to the control of Plasmodium falciparum malaria. Recently, resistance to the artemisinin combination therapy partner drug piperaquine has been observed in multiple locations across Southeast Asia. Genetic studies have identified single nucleotide polymorphisms as well as copy number variations in the plasmepsin 2 and plasmepsin 3 genes, which encode haemoglobin-degrading proteases that associate with clinical and in vitro piperaquine resistance.
RESULTS
To accurately and quickly determine the presence of copy number variations in the plasmepsin 2/3 genes in field isolates, this study developed a quantitative PCR assay using TaqMan probes. Copy number estimates were validated using a separate SYBR green-based quantitative PCR assay as well as a novel PCR-based breakpoint assay to detect the hybrid gene product. Field samples from 2012 to 2015 across three sites in Cambodia were tested using DNA extracted from dried blood spots and whole blood to monitor the extent of plasmepsin 2/3 gene amplifications, as well as amplifications in the multidrug resistance transporter 1 gene (pfmdr1), a marker of mefloquine resistance. This study found high concordance across all methods of copy number detection. For samples derived from dried blood spots, a success rate greater than 80% was found in each assay, with more recent samples performing better. Evidence of extensive plasmepsin 2/3 copy number amplifications was observed in Pursat (94%, 2015) (Western Cambodia) and Preah Vihear (87%, 2014) (Northern Cambodia), and lower levels in Ratanakiri (16%, 2014) (Eastern Cambodia). A shift was observed from two copies of plasmepsin 2 in Pursat in 2013 to three copies in 2014-2015 (25% to 64%). Pfmdr1 amplifications were absent in all samples from Preah Vihear and Ratanakiri in 2014 and absent in Pursat in 2015.
CONCLUSIONS
The multiplex TaqMan assay is a robust tool for monitoring both plasmepsin 2/3 and pfmdr1 copy number variations in field isolates, and the SYBR-green and breakpoint assays are useful for monitoring plasmepsin 2/3 amplifications. This study shows increasing levels of plasmepsin 2 copy numbers across Cambodia from 2012 to 2015 and a complete reversion of multicopy pfmdr1 parasites to single copy parasites in all study locations.
背景
东南亚长期以来一直被认为是抗药性疟原虫的中心，它继续给恶性疟原虫疟疾的控制带来新的挑战。最近，在东南亚多个地区发现了对青蒿素联合疗法的联合用药哌喹的耐药性。遗传研究已经确定了疟原蛋白酶 2 和疟原蛋白酶 3 基因中的单核苷酸多态性和拷贝数变异，这些基因编码与临床和体外哌喹耐药性相关的血红蛋白降解蛋白酶。
结果
为了准确快速地确定现场分离株中疟原蛋白酶 2/3 基因的拷贝数变异，本研究使用 TaqMan 探针开发了一种定量 PCR 检测方法。通过单独的 SYBR 绿色定量 PCR 检测以及新型 PCR 断点检测来验证拷贝数估计，以检测杂交基因产物。使用从 2012 年至 2015 年在柬埔寨三个地点采集的干血斑和全血提取的 DNA 来检测现场样本，以监测疟原蛋白酶 2/3 基因扩增的程度，以及多药耐药转运蛋白 1 基因 (pfmdr1) 的扩增情况，这是氯喹耐药的标志。本研究发现，所有拷贝数检测方法的一致性都很高。对于源自干血斑的样本，每个检测方法的成功率均超过 80%，最近的样本效果更好。在柏威夏（94%，2015 年）（柬埔寨西部）和腊塔纳基里（16%，2014 年）（柬埔寨东部）发现了广泛的疟原蛋白酶 2/3 拷贝数扩增证据，而在拉达那基里（87%，2014 年）（柬埔寨北部）则较低。在 2013 年，柏威夏的疟原蛋白酶 2 有两个拷贝，到 2014-2015 年增加到三个拷贝（25%到 64%）。在 2014 年，腊塔纳基里和柏威夏的所有样本均未发现 pfmdr1 扩增，2015 年在柏威夏也未发现。
结论
多重 TaqMan 检测法是监测现场分离株中疟原蛋白酶 2/3 和 pfmdr1 拷贝数变化的可靠工具，SYBR-绿色和断点检测法可用于监测疟原蛋白酶 2/3 的扩增。本研究表明，2012 年至 2015 年期间，柬埔寨的疟原蛋白酶 2 拷贝数不断增加，所有研究地点的多拷贝 pfmdr1 寄生虫完全恢复为单拷贝寄生虫。
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