Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia.
Malaria and NTDs Research Team, Bacterial, Parasitic, and Zoonotic Diseases Research Directorate, Ethiopian Public Health Institute, Addis Ababa, Ethiopia.
Malar J. 2024 Apr 17;23(1):108. doi: 10.1186/s12936-024-04904-3.
Rapid diagnostic tests (RDTs) play a significant role in expanding case management in peripheral healthcare systems. Histidine-rich protein-2 (HRP2) antigen detection RDTs are predominantly used to diagnose Plasmodium falciparum infection. However, the evolution and spread of P. falciparum parasite strains with deleted hrp2/3 genes, causing false-negative results, have been reported. This study assessed the diagnostic performance of HRP2-detecting RDTs for P. falciparum cases and the prevalence of pfhrp2/3 deletions among symptomatic patients seeking malaria diagnosis at selected health facilities in southern Ethiopia.
A multi-health facilities-based cross-sectional study was conducted on self-presenting febrile patients seeking treatment in southern Ethiopia from July to September 2022. A purposive sampling strategy was used to enroll patients with microscopically confirmed P. falciparum infections. A capillary blood sample was obtained to prepare a blood film for microscopy and a RDT using the SD Bioline™ Malaria Pf/Pv Test. Dried blood spot samples were collected for further molecular analysis. DNA was extracted using gene aid kits and amplification was performed using nested PCR assay. Exon 2 of hrp2 and hrp3, which are the main protein-coding regions, was used to confirm its deletion. The diagnostic performance of RDT was evaluated using PCR as the gold standard test for P. falciparum infections.
Of 279 P. falciparum PCR-confirmed samples, 249 (89.2%) had successful msp-2 amplification, which was then genotyped for hrp2/3 gene deletions. The study revealed that pfhrp2/3 deletions were common in all health centres, and it was estimated that 144 patients (57.8%) across all health facilities had pfhrp2/3 deletions, leading to false-negative PfHRP2 RDT results. Deletions spanning exon 2 of hrp2, exon 2 of hrp3, and double deletions (hrp2/3) accounted for 68 (27.3%), 76 (30.5%), and 33 (13.2%) of cases, respectively. The study findings revealed the prevalence of P. falciparum parasites lacking a single pfhrp2-/3-gene and that both genes varied across the study sites. This study also showed that the sensitivity of the SD Bioline PfHRP2-RDT test was 76.5% when PCR was used as the reference test.
This study confirmed the existence of widespread pfhrp2/3- gene deletions, and their magnitude exceeded the WHO-recommended threshold (> 5%). False-negative RDT results resulting from deletions in Pfhrp2/3- affect a country's attempts at malaria control and elimination. Therefore, the adoption of non-HRP2-based RDTs as an alternative measure is required to avoid the consequences associated with the continued use of HRP-2-based RDTs, in the study area in particular and in Ethiopia in general.
快速诊断检测（RDT）在扩大周边医疗保健系统中的病例管理方面发挥着重要作用。富含组氨酸蛋白-2（HRP2）抗原检测 RDT 主要用于诊断恶性疟原虫感染。然而，已经报道了恶性疟原虫寄生虫株中 HRP2/3 基因缺失导致假阴性结果的进化和传播。本研究评估了 HRP2 检测 RDT 在诊断恶性疟原虫病例中的诊断性能，以及在埃塞俄比亚南部选定卫生设施就诊的有症状患者中 pfhrp2/3 缺失的流行情况。
这是一项在埃塞俄比亚南部 2022 年 7 月至 9 月期间于多个医疗机构开展的基于横断面的研究。采用目的抽样策略招募经显微镜确认患有恶性疟原虫感染的有发热症状的患者。采集末梢血样制备血片进行显微镜检查和使用 SD Bioline™ 疟疾 Pf/Pv 检测的 RDT。采集干血斑样本进行进一步的分子分析。使用基因 aid 试剂盒提取 DNA，并使用巢式 PCR 检测进行扩增。扩增目的是确认 HRP2 和 HRP3 的外显子 2 缺失，这是主要的蛋白编码区。使用 PCR 作为恶性疟原虫感染的金标准检测，评估 RDT 的诊断性能。
在 279 份经恶性疟原虫 PCR 确认的样本中，有 249 份（89.2%）成功扩增 msp-2，然后对其进行 HRP2/3 基因缺失的基因分型。研究表明，所有卫生中心都普遍存在 pfhrp2/3 缺失，估计所有卫生设施共有 144 名（57.8%）患者存在 pfhrp2/3 缺失，导致 PfHRP2 RDT 结果假阴性。HRP2 外显子 2 缺失、HRP3 外显子 2 缺失和双缺失（hrp2/3）分别占 68 例（27.3%）、76 例（30.5%）和 33 例（13.2%）。研究结果显示，恶性疟原虫寄生虫缺乏单个 pfhrp2-/3-基因的流行情况以及在研究地点之间存在的双基因缺失情况。本研究还表明，当使用 PCR 作为参考测试时，SD Bioline PfHRP2-RDT 测试的灵敏度为 76.5%。
本研究证实了 pfhrp2/3-基因缺失的广泛存在，其程度超过了世界卫生组织建议的阈值（>5%）。由于 Pfhrp2/3-缺失导致的 RDT 假阴性结果会影响一个国家控制和消除疟疾的努力。因此，需要采用非 HRP2 基 RDT 作为替代措施，避免因继续使用 HRP-2 基 RDT 而产生的后果，特别是在研究区域，以及在整个埃塞俄比亚。
