Infectious Diseases Research Collaboration, Kampala, Uganda.
Department of Pathology, College of Health Science, Makerere University Kampala, Kampala, Uganda.
Malar J. 2021 Jan 2;20(1):4. doi: 10.1186/s12936-020-03547-4.
Rapid diagnostic tests (RDTs) play a key role in malaria case management. The most widely used RDT identifies Plasmodium falciparum based on immunochromatographic recognition of P. falciparum histidine-rich protein 2 (PfHRP2). Deletion of the paralogous pfhrp2 and pfhrp3 genes leads to false-negative PfHRP2-based RDTs, and has been reported in P. falciparum infections from South America and Africa. However, identification of pfhrp2/pfhrp3 deletions has usually been based only on failure to amplify these genes using PCR, without confirmation based on PfHRP2 protein expression, and understanding of the true prevalence of deletions is incomplete.
Deletions of pfhrp2/pfhrp3 in blood samples were investigated from cross-sectional surveys in 2012-13 in three regions of varied malaria transmission intensity in Uganda. Samples with positive Giemsa-stained thick blood smears, but negative PfHRP2-based RDTs were evaluated by PCR amplification of conserved subunit ribosomal DNA for Plasmodium species, PCR amplification of pfhrp2 and pfhrp3 genes to identify deletions, and bead-based immunoassays for expression of PfHRP2.
Of 3516 samples collected in cross-sectional surveys, 1493 (42.5%) had positive blood smears, of which 96 (6.4%) were RDT-negative. Of these 96 RDT-negative samples, P. falciparum DNA was identified by PCR in 56 (58%) and only non-falciparum plasmodial DNA in 40 (42%). In all 56 P. falciparum-positive samples there was a failure to amplify pfhrp2 or pfhrp3: in 25 (45%) pfhrp2 was not amplified, in 39 (70%) pfhrp3 was not amplified, and in 19 (34%) neither gene was amplified. For the 39 P. falciparum-positive, RDT-negative samples available for analysis of protein expression, PfHRP2 was not identified by immunoassay in only four samples (10.3%); these four samples all had failure to amplify both pfhrp2 and pfhrp3 by PCR. Thus, only four of 96 (4.2%) smear-positive, RDT-negative samples had P. falciparum infections with deletion of pfhrp2 and pfhrp3 confirmed by failure to amplify the genes by PCR and lack of expression of PfHRP2 demonstrated by immunoassay.
False negative RDTs were uncommon. Deletions in pfhrp2 and pfhrp3 explained some of these false negatives, but most false negatives were not due to deletion of the pfhrp2 and pfhrp3 genes.
快速诊断检测（RDT）在疟疾病例管理中起着关键作用。最广泛使用的 RDT 通过免疫层析法识别恶性疟原虫裂殖子表面蛋白 2（PfHRP2）来识别恶性疟原虫。PfHRP2 和 PfHRP3 基因的同源基因缺失会导致基于 PfHRP2 的 RDT 出现假阴性，这种情况已在南美洲和非洲的恶性疟原虫感染中报道过。然而，pfhrp2/pfhrp3 缺失的鉴定通常仅基于 PCR 未能扩增这些基因，而没有基于 PfHRP2 蛋白表达的确认，并且对缺失的真实流行率的了解并不完整。
在乌干达三个疟疾传播强度不同的地区，于 2012-13 年进行了横断面调查，对血液样本中的 pfhrp2/pfhrp3 缺失情况进行了调查。对吉姆萨染色厚血涂片阳性但基于 PfHRP2 的 RDT 阴性的样本，通过聚合酶链反应（PCR）扩增种系核糖体 DNA 以鉴定所有疟原虫物种，PCR 扩增 pfhrp2 和 pfhrp3 基因以鉴定缺失情况，以及通过基于珠的免疫测定法检测 PfHRP2 的表达情况。
在横断面调查中采集了 3516 个样本，其中 1493 个（42.5%）血涂片阳性，其中 96 个（6.4%）RDT 阴性。在这 96 个 RDT 阴性样本中，通过 PCR 鉴定出 56 个（58%）有恶性疟原虫 DNA，40 个（42%）只有非恶性疟原虫 DNA。在所有 56 个恶性疟原虫阳性样本中，pfhrp2 或 pfhrp3 均未能扩增：在 25 个（45%）样本中，pfhrp2 未能扩增，在 39 个（70%）样本中，pfhrp3 未能扩增，在 19 个（34%）样本中，这两个基因均未能扩增。在可用于分析蛋白表达的 39 个 PfHRP2 阳性、RDT 阴性样本中，只有 4 个样本（10.3%）无法通过免疫测定法鉴定 PfHRP2；这 4 个样本均通过 PCR 未能扩增 pfhrp2 和 pfhrp3。因此，在 96 个血涂片阳性、RDT 阴性样本中，仅有 4 个（4.2%）通过 PCR 未能扩增基因且免疫测定法未检测到 PfHRP2 表达证实存在 PfHRP2 和 PfHRP3 基因缺失的恶性疟原虫感染。
假阴性 RDT 并不常见。pfhrp2 和 pfhrp3 的缺失解释了其中一些假阴性，但大多数假阴性并非由于 pfhrp2 和 pfhrp3 基因缺失所致。
