Malaria Branch, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA, 30329, USA.
The CDC Foundation, 600 Peachtree Street NE, Suite 1000, Atlanta, GA, 30308, USA.
Malar J. 2021 Apr 29;20(1):207. doi: 10.1186/s12936-021-03739-6.
The Plasmodium falciparum antigen histidine rich protein 2 (HRP2) is a preferred target for malaria rapid diagnostic tests (RDTs) because of its abundant production by the parasite and thermal stability. As a result, a majority of RDTs procured globally target this antigen. However, previous reports from South America and recent reports from sub-Saharan Africa and Asia indicate that certain P. falciparum parasites have deletions of the gene coding for HRP2. The HRP2 antigen is paralogous to another P. falciparum antigen HRP3 and some antibodies to HRP2 cross-react with HRP3. Multiple parasites have been described with deletions of one or both hrp2 and hrp3 genes. It is unclear how the various combinations of hrp2 and hrp3 deletion genotypes affect clinical sensitivity of HRP2-based RDTs.
Cross-reactivity between HRP2 and HRP3 was tested on malaria RDTs using culture-adapted P. falciparum parasites with both hrp2 and hrp3 intact or with one or both genes deleted. Ten-fold serial dilutions of four culture-adapted P. falciparum parasites [3D7 (hrp2+/hrp3+), Dd2 (hrp2-/hrp3+), HB3 (hrp2+/hrp3-) and 3BD5 (hrp2-/hrp3-)] ranging from 100,000 to 0.01 parasites/µL were prepared. HRP2, Plasmodium lactate dehydrogenase (pLDH) and aldolase concentrations were determined for the diluted samples using a multiplex bead assay. The samples were subsequently tested on three RDT products designed to detect P. falciparum by HRP2 alone or in combination with pLDH.
At parasite densities of approximately 1000 parasites/µL, parasites that expressed either hrp2 or hrp3 were detected by all three RDTs. Multiplex based antigen measurement using HRP2- conjugated beads demonstrated higher antigen concentration when both hrp2 and hrp3 genes were intact (3D7 parasites, 47.9 ng/ml) compared to HB3 (3.02 ng/mL) and Dd2 (0.20 ng/mL) strains that had one gene deleted. 3D7 at 10 parasites/µL (0.45 ng/mL) was reactive on all three RDT products whereas none of the other parasites were reactive at that density.
Above a certain antigen threshold, HRP3 cross-reactivity on HRP2-based RDTs is sufficient to mask the effects of deletions of hrp2 only. Studies of hrp2 deletion and its effects on HRP2-based RDTs must be studied alongside hrp3 deletions and include clinical sample reactivity on HRP2-based tests.
恶性疟原虫抗原裂殖子表面蛋白 2（HRP2）是疟疾快速诊断检测（RDT）的首选靶标，因为它由寄生虫大量产生且热稳定性高。因此，全球大多数 RDT 都针对这种抗原。然而，来自南美洲的先前报告以及来自撒哈拉以南非洲和亚洲的最近报告表明，某些恶性疟原虫寄生虫存在 HRP2 编码基因的缺失。HRP2 抗原与另一种恶性疟原虫抗原 HRP3 是旁系同源的，一些针对 HRP2 的抗体与 HRP3 发生交叉反应。已经描述了多种缺失一个或两个 hrp2 和 hrp3 基因的寄生虫。尚不清楚 HRP2 缺失基因型的各种组合如何影响基于 HRP2 的 RDT 的临床敏感性。
使用具有完整的 hrp2 和 hrp3 或缺失一个或两个基因的培养适应恶性疟原虫寄生虫，在疟疾 RDT 上测试 HRP2 和 HRP3 之间的交叉反应性。从 100,000 到 0.01 个寄生虫/µL ，制备了四个培养适应的恶性疟原虫寄生虫[3D7（hrp2+/hrp3+）、Dd2（hrp2-/hrp3+）、HB3（hrp2+/hrp3-）和 3BD5（hrp2-/hrp3-）]的十倍系列稀释液。使用多重珠粒测定法测定稀释样品中的 HRP2、疟原虫乳酸脱氢酶（pLDH）和醛缩酶浓度。随后将这些样品用于三种设计用于通过 HRP2 单独或与 pLDH 组合检测恶性疟原虫的 RDT 产品上进行检测。
在大约 1000 个寄生虫/µL 的寄生虫密度下，所有三种 RDT 都可以检测到表达 HRP2 或 HRP3 的寄生虫。使用 HRP2 偶联珠粒进行的基于多重的抗原测量显示，当两个 hrp2 和 hrp3 基因都完整时（3D7 寄生虫，47.9 ng/ml），与 HB3（3.02 ng/mL）和 Dd2（0.20 ng/mL）相比，抗原浓度更高，这两种寄生虫均缺失一个基因。在 10 个寄生虫/µL（0.45 ng/mL）时，所有三种 RDT 产品均检测到 3D7，但其他寄生虫均未在该密度下反应。
在一定的抗原阈值以上，基于 HRP2 的 RDT 上的 HRP3 交叉反应性足以掩盖仅缺失 hrp2 的影响。必须对 HRP2 缺失及其对基于 HRP2 的 RDT 的影响进行研究，同时还要对 HRP3 缺失进行研究，并包括基于 HRP2 的检测的临床样本反应性。
