False-negative results for Plasmodium falciparum histidine-rich protein (HRP) 2–based rapid diagnostic tests (RDTs) are increasing in Eritrea. We investigated HRP gene 2/3 (pfhrp2/pfhrp3) status in 50 infected patients at 2 hospitals. We showed that 80.8% (21/26) of patients at Ghindae Hospital and 41.7% (10/24) at Massawa Hospital were infected with pfhrp2-negative parasites and 92.3% (24/26) of patients at Ghindae Hospital and 70.8% (17/24) at Massawa Hospital were infected with pfhrp3-negative parasites. Parasite densities between pfhrp2-positive and pfhrp2-negative patients were comparable. All pfhrp2-negative samples had no detectable HRP2/3 antigen and showed negative results for HRP2-based RDTs. pfhrp2-negative parasites were genetically less diverse and formed 2 clusters with no close relationships to parasites from Peru. These parasites probably emerged independently by selection in Eritrea. High prevalence of pfhrp2-negative parasites caused a high rate of false-negative results for RDTs. Determining prevalence of pfhrp2-negative parasites is urgently needed in neighboring countries to assist case management policies.

Background: Histidine-rich protein-2 (HRP2)-based rapid diagnostic tests (RDTs) are the only RDTs recommended for malaria diagnosis in Uganda. However, the emergence of Plasmodium falciparum histidine rich protein 2 and 3 (pfhrp2 and pfhrp3) gene deletions threatens their usefulness as malaria diagnostic and surveillance tools. The pfhrp2 and pfhrp3 gene deletions surveillance was conducted in P. falciparum parasite populations in Uganda. Methods: Three-hundred (n = 300) P. falciparum isolates collected from cross-sectional malaria surveys in symptomatic individuals in 48 districts of eastern and western Uganda were analysed for the presence of pfhrp2 and pfhrp3 genes. Presence of parasite DNA was confirmed by PCR amplification of the 18s rRNA gene, msp1 and msp2 single copy genes. Presence or absence of deletions was confirmed by amplification of exon1 and exon2 of pfhrp2 and pfhrp3 using gene specific PCR. Results: Overall, pfhrp2 and pfhrp3 gene deletions were detected in 29/300 (9.7%, 95% CI 6.6-13.6%) parasite isolates. The pfhrp2 gene was deleted in 10/300 (3.3%, 95% CI 1.6-6.0%) isolates, pfhrp3 in 9/300 (3.0%, 95% CI 1.4-5.6%) while both pfhrp2 and pfhrp3 were deleted in 10/300 (3.3%, 95% CI 1.6-6.0%) parasite isolates. Proportion of pfhrp2/3 deletions was higher in the eastern 14.7% (95% CI 9.7-20.0%) compared to the western region 3.1% (95% CI 0.8-7.7%), p = 0.001. Geographical location was associated with gene deletions aOR 6.25 (2.02-23.55), p = 0.003. Conclusions: This is the first large-scale survey reporting the presence of pfhrp2/3 gene deletions in P. falciparum isolates in Uganda. Roll out of RDTs for malaria diagnosis should take into consideration the existence of pfhrp2/3 gene deletions particularly in areas where they were detected. Periodic pfhrp2/3 surveys are recommended to inform future decisions for deployment of alternative RDTs.

Eritrea was the first African country to complete a nationwide switch in 2016 away from HRP2-based RDTs due to high rates of false-negative RDT results caused by Plasmodium falciparum parasites lacking hrp2/hrp3 genes. A cross-sectional survey was conducted during 2019 enrolling symptomatic malaria patients from nine health facilities across three zones consecutively to investigate the epidemiology of P. falciparum lacking hrp2/3 after the RDT switch. Molecular analyses of 715 samples revealed the overall prevalence of hrp2-, hrp3-, and dual hrp2/3-deleted parasites as 9.4% (95%CI 7.4–11.7%), 41.7% (95% CI 38.1–45.3%) and 7.6% (95% CI 5.8–9.7%), respectively. The prevalence of hrp2- and hrp3-deletion is heterogeneous within and between zones: highest in Anseba (27.1% and 57.9%), followed by Gash Barka (6.4% and 37.9%) and Debub zone (5.2% and 43.8%). hrp2/3-deleted parasites have multiple diverse haplotypes, with many shared or connected among parasites of different hrp2/3 status, indicating mutant parasites have likely evolved from multiple and local parasite genetic backgrounds. The findings show although prevalence of hrp2/3-deleted parasites is lower 2 years after RDT switching, HRP2-based RDTs remain unsuitable for malaria diagnosis in Eritrea. Continued surveillance of hrp2/3-deleted parasites in Eritrea and neighbouring countries is required to monitor the trend.

Background Plasmodium falciparum histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDTs) are exclusively recommended for malaria diagnosis in Uganda; however, their functionality can be affected by parasite-related factors that have not been investigated in field settings. Methods Using a cross-sectional design, we analysed 219 RDT-/microscopy+ and 140 RDT+/microscopy+ dried blood spots obtained from symptomatic children aged 2–10 years from 48 districts in Uganda between 2017 and 2019. We aimed to investigate parasite-related factors contributing to false RDT results by molecular characterization of parasite isolates. ArcGIS software was used to map the geographical distribution of parasites. Statistical analysis was performed using chi-square or Fisher’s exact tests, with P ≤ 0.05 indicating significance. Odds ratios (ORs) were used to assess associations, while logistic regression was performed to explore possible factors associated with false RDT results. Results The presence of parasite DNA was confirmed in 92.5% (332/359) of the blood samples. The levels of agreement between the HRP2 RDT and PCR assay results in the (RDT+/microscopy+) and (RDT-/microscopy+) sample subsets were 97.8% (137/140) and 10.9% (24/219), respectively. Factors associated with false-negative RDT results in the (RDT-/microscopy+) samples were parasite density (<1,000/μl), pfhrp2/3 gene deletion and non-P. falciparum species (aOR 2.65, 95% CI: 1.62–4.38, P = 0.001; aOR 4.4, 95% CI 1.72–13.66, P = 0.004; and aOR 18.65, 95% CI: 5.3–38.7, P = 0.001, respectively). Overall, gene deletion and non-P. falciparum species contributed to 12.3% (24/195) and 19.0% (37/195) of false-negative RDT results, respectively. Of the false-negative RDTs results, 80.0% (156/195) were from subjects with low-density infections (< 25 parasites per 200 WBCs or <1,000/μl). Conclusion This is the first evaluation and report of the contributions of pfhrp2/3 gene deletion, non-P. falciparum species, and low-density infections to false-negative RDT results under field conditions in Uganda. In view of these findings, the use of HRP2 RDTs should be reconsidered; possibly, switching to combination RDTs that target alternative antigens, particularly in affected areas, may be beneficial. Future evaluations should consider larger and more representative surveys covering other regions of Uganda.

Deletion of histidine-rich protein genes pfhrp2/3 in Plasmodium falciparum causes infections to go undetected by HRP2-based malaria rapid diagnostic tests. We analyzed P. falciparum malaria cases imported to Australia (n = 210, collected 2010–2018) for their pfhrp2/3 status. We detected gene deletions in patients from 12 of 25 countries. We found >10% pfhrp2- deletion levels in those from Nigeria (13.3%, n = 30), Sudan (11.2%, n = 39), and South Sudan (17.7%, n = 17) and low levels of pfhrp3 deletion from Sudan (3.6%) and South Sudan (5.9%). No parasites with pfhrp2/3 double deletions were detected. Microsatellite typing of parasites from Nigeria, Sudan, and South Sudan revealed low relatedness among gene-deleted parasites, indicating independent emergences. The gene deletion proportions signify a risk of false-negative HRP2-RDT results. This study’s findings warrant surveillance to determine whether the prevalence of gene-deleted parasites justifies switching malaria rapid diagnostic tests in Nigeria, Sudan, and South Sudan.