Dept. of Immunology, Noguchi Memorial Institute of Medical Research, University of Ghana, Accra, Ghana.
Dept. of Epidemiology, Noguchi Memorial Institute of Medical Research, University of Ghana, Accra, Ghana.
PLoS One. 2020 Sep 4;15(9):e0238749. doi: 10.1371/journal.pone.0238749. eCollection 2020.
False-negative malaria rapid diagnostic test (RDT) results amongst symptomatic malaria patients are detrimental as they could lead to ineffective malaria case management. This study determined the nationwide contribution of parasites with Pfhrp2 and Pfhrp 3 gene deletions to false negative malaria RDT results in Ghana.
This was a cross sectional study where whole blood (~2 ml) was collected from patients presenting with malaria symptoms at 100 health facilities in all the regions in Ghana from May to August 2018. An aliquot of the blood was used to prepare thin and thick blood smears, filter paper blood spots (DBS) and spot a PfHRP 2 RDT kit. The remaining blood was separated into plasma and blood cells and stored at -20°C. Plasmodium parasite density and species identity was estimated from the blood smears. Plasmodium falciparum specific 18S rRNA PCR, merozoite surface protein (msp 1) and glutamate rich protein (glurp) gene PCR were used to identify P. falciparum positive samples, which were subjected to Pfhrp 2/3 exon1-2 and exon2 genotyping.
Of the 2,860 microscopically P. falciparum positive patients analyzed, 134 (4.69%) had false negative P. falciparum specific RDT results. Samples for PCR analysis was available for 127 of the false negative patients, and the analysis identified 116 (91.3%) as positive for P. falciparum. Only 58.1% (79/116) of the false negative RDT samples tested positive by msp 1 and glurp PCR. Genotyping of exon 1-2 and exon 2 of the Pfhrp 2 gene identified 12.9% (10/79) and 39.5% (31/79) of samples respectively to have deletions. Genotyping exon 1-2 and exon 2 of the Pfhrp 3 gene identified 15.2% (12/79) and 40.5% (32/79) of samples respectively to have deletions. Only 5% (4/79) of the false negative samples had deletions in both exon 1-2 and exon 2 of the Pfhrp 2 gene. Out of the 49 samples that tested positive for aldolase by luminex, 32.6% (16/49) and) had deletions in Pfhrp 2 exon 2 and 2% (1/49) had deletions in both exon 2 and exon 1-2 of the Pfhrp 2 gene.
The low prevalence of false negative RDT test results provides assurance that PfHRP 2 based malaria RDT kits remain effective in diagnosing symptomatic malaria patients across all the Regions of Ghana. Although there was a low prevalence of parasites with deletions in exon 2 and exon 1-2 of the Pfhrp 2 gene the prevalence of parasites with deletions in Pfhrp 2 exon 2 was about a third of the false negative RDT results. The need to ensure rapid, accurate and reliable malaria diagnosis requires continuous surveillance of parasites with Pfhrp 2 gene deletions.
在有症状的疟疾患者中，疟原虫快速诊断检测（RDT）出现假阴性结果是有害的，因为这可能导致疟疾病例管理无效。本研究旨在确定加纳全国范围内 Pfhrp2 和 Pfhrp3 基因缺失的寄生虫对疟疾 RDT 假阴性结果的贡献。
这是一项横断面研究，于 2018 年 5 月至 8 月期间，从加纳所有地区的 100 个卫生机构中出现疟疾症状的患者中采集约 2ml 全血。血液的一部分用于制备薄血涂片和厚血涂片、滤纸血斑（DBS）和 PfHRP2 RDT 试剂盒。其余血液分离为血浆和血细胞，并储存在-20°C。从血涂片估计疟原虫密度和种属身份。使用疟原虫 18S rRNA PCR、裂殖子表面蛋白（msp1）和谷氨酸丰富蛋白（glurp）基因 PCR 来鉴定 Pfalciparum 阳性样本，并对 Pfhrp2/3 外显子 1-2 和外显子 2 进行基因分型。
在分析的 2860 例经显微镜确认的 Pfalciparum 阳性患者中，有 134 例（4.69%）出现 Pfalciparum 特异性 RDT 假阴性结果。有 127 例假阴性患者可进行 PCR 分析，其中 116 例（91.3%）被鉴定为 Pfalciparum 阳性。只有 58.1%（79/116）的假阴性 RDT 样本通过 msp1 和 glurp PCR 检测呈阳性。Pfhrp2 基因外显子 1-2 和外显子 2 的基因分型分别确定了 12.9%（10/79）和 39.5%（31/79）的样本有缺失。Pfhrp3 基因外显子 1-2 和外显子 2 的基因分型分别确定了 15.2%（12/79）和 40.5%（32/79）的样本有缺失。只有 5%（4/79）的假阴性样本在 Pfhrp2 基因的外显子 1-2 中有缺失。在 49 例 aldolase 检测呈阳性的样本中，有 32.6%（16/49）和 2%（1/49）的样本在 Pfhrp2 外显子 2 中有缺失，有 2%（1/49）的样本在 Pfhrp2 基因的外显子 2 和外显子 1-2 中有缺失。
假阴性 RDT 检测结果的低患病率保证了基于 PfHRP2 的疟疾 RDT 试剂盒在加纳所有地区诊断有症状的疟疾患者仍然有效。尽管 Pfhrp2 基因外显子 2 和外显子 1-2 缺失的寄生虫患病率较低，但 Pfhrp2 外显子 2 缺失的寄生虫患病率约占假阴性 RDT 结果的三分之一。为确保疟疾快速、准确和可靠诊断，需要持续监测 Pfhrp2 基因缺失的寄生虫。
