Are HRP2/3 deletions silently crippling malaria rapid diagnostic tests?

Search strategy and data sources

Literature searches were done in the electronic databases: PubMed, Google Scholar and Scopus, including publications from 2010 to 2025. The search string used was:”pfhrp2” OR “pfhrp3” OR “HRP2” OR “HRP3” OR “hrp2/3” AND “malaria” OR “Plasmodium falciparum” AND deletion OR “deletion rate” OR prevalence OR “RDT failure”. Additionally, sources were identified through the reference lists of relevant articles and reviews. Studies with full text available in English were included.

Study selection

World Health Organization (WHO) technical documents and surveillance guidelines relevant to HRP2 diagnostics were also included. Out of 148 articles retrieved from the various databases, a total of 120 articles were excluded based on the criteria for article selection for the review. In total, 28 peer-reviewed research articles and WHO Global Strategy document were selected after initial screening by title and abstract. Article selection process has been depicted in the Figure.

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Figure.

Articles selection process. WHO, World Health Organization; RDT, rapid diagnostic test; HRP, histidine rich protein.

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Study type and place of study

Original research studies that reported molecular detection of pfhrp2 and/or pfhrp3 deletions in P. falciparum, were included in this review. The occurrence, detection, consequences, and regional distribution of pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum are the main topics of 28 peer-reviewed articles that make up this review of the literature. The investigations came from South America, Asia, and Africa, three continents where malaria is endemic. To offer a strategic perspective, WHO’s Global Technical Strategy for Malaria and associated policy documents were also included.

Study characteristics

South America

South America has also experienced a notable prevalence of deletions. This pivotal study in the Peruvian Amazon revealed an alarmingly high prevalence of pfhrp2/3 gene deletions. Among 148 P. falciparum samples, 41% (61/148) lacked the pfhrp2 gene, 70% (103/148) lacked pfhrp3, and 21.6% (32/148) had dual pfhrp2/3 deletions. This widespread absence of target genes severely compromises HRP2-based RDTs, leading to substantial false negatives and underscoring an urgent need for alternative diagnostic strategies in the region.²⁸

Brazil’s Middle Rio Negro region recorded up to 18.3% of double deletions. This study found a high prevalence of P. falciparum with pfhrp2 (34.2%), pfhrp3 (23.2%), and double pfhrp2/3 (18.3%) gene deletions, which posed a significant threat to accuracy of HRP2-based RDT.²⁹

These findings indicate that HRP2 deletions are not confined to Africa and highlight the global nature of the challenge. False negatives resulting from HRP2 deletions undermine the “test-and-treat” strategy central to malaria control. Patients testing negative are unlikely to receive treatment, increasing the risk of complications and community-level transmission. In Eritrea and Ethiopia, this diagnostic failure contributed to delays in treatment and increased malaria burden.¹

Alternative diagnostic methods

Molecular diagnostic techniques

A study was done focused on developing and validating a high-throughput digital PCR (dPCR) method for accurate pfhrp2/3 gene deletion typing. The dPCR assay demonstrated superior sensitivity, capable of detecting and quantifying deletions in samples with very low parasite densities, specifically down to 0.33 parasites/µL. This methodological advancement is crucial for enhancing the efficiency and precision of large-scale surveillance efforts for pfhrp2/3 deletions. By enabling better monitoring, this dPCR tool empowers public health programs to more effectively track the spread of deletion mutants and manage the implications for HRP2-based RDT efficacy worldwide.³³

A 2-yr study done in JIPMER, Puducherry found that 4 out of 13 (30.7%) P. falciparum cases were RDT-negative but microscopy-positive and they were confirmed to lack pfhrp2 and pfhrp3 genes by PCR. These deletions highlight the risk of false-negative HRP2-RDTs and the need for routine molecular surveillance.³⁴

The 2024 WHO’s Surveillance Template Protocol for pfhrp2/pfhrp3 Gene Deletions highlights an approach to monitor Plasmodium falciparum gene deletions that affect HRP2-based RDTs. It suggests a tiered diagnostic strategy, starting with PCR screening, followed by digital droplet PCR (ddPCR) or whole-genome sequencing (WGS) for confirmation and lineage analysis. If ≥5% of symptomatic cases show false-negative HRP2-RDT results due to deletions, change to non-HRP2-based diagnostics is advised. The protocol also put emphasis on sample biobanking for long-term surveillance and research. This framework supports timely policy decisions to maintain effective malaria diagnosis.³⁵

Policy and surveillance

The WHO encourages national malaria control programs to incorporate regional deletion data into diagnostic procurement decisions and testing algorithms.^1,36

Future strategies must include robust global surveillance networks that share real-time data on deletion prevalence. WHO’s call for interactive dashboards and global mapping tools aims to inform diagnostic policy shifts dynamically.³⁶ There is also a pressing need for the development of multiplex RDTs that detect multiple antigens or rely on HRP2-independent markers.³⁷

A study done in Ethiopia shows emerging resistance of P. Falciparum to both artemisinin-based treatments and HRP2 RDTs. A key finding was 8.0% of malaria cases carried the kelch13 (K13) 622I mutation, which is linked to artemisinin in partial resistance. This co-occurrence threatens “test-and-treat” strategies, calling for urgent, close monitoring and alternative diagnostic/treatment strategies for effective malaria control efforts.³⁸

Studies between 2010 and 2025 reveal a progressive expansion of pfhrp2/3 deletions across many malaria-endemic regions. Initially (2010–2014) reports were mainly restricted to South America, especially the Peruvian Amazon and some parts of Brazil, where deletion prevalence often exceeded 20%. From 2015, several African countries reported prevalence exceeded the WHO 5% threshold which included Eritrea, Ethiopia, Sudan, South Sudan, Nigeria, and Tanzania. Recent data from 2020–2025 indicate steady and in some areas, very high prevalence, which included southern Ethiopia reporting deletion rates crossing 50%.^21,28

More research is required in under-reported regions such as Central Africa and Southeast Asia to understand the true burden of HRP2 deletions. Additionally, integrating molecular diagnostics into routine case surveillance and bio banking clinical samples can support real-time response strategies. WHO recommends that surveillance data on pfhrp2 deletions be used to guide timely switching to non-HRP2–based RDTs when HRP2-based diagnostics produce false-negative results due to gene deletions. The genetic diversity of HRP2, particularly in border regions such as the China-Myanmar area, suggests that even in the absence of deletions, antigenic variability may compromise test sensitivity and warrants inclusion in ongoing surveillance efforts.³⁷

Integration with WHO’s global technical strategy for malaria 2016–2030

Universal access to accurate diagnosis as a basis of malaria elimination is emphasized in The WHO Global Technical Strategy for Malaria 2016–2030. The rise of pfhrp2/3 deletions affects HRP2-based RDTs, leading to false negative results, delayed management, and weakened surveillance systems, thereby threatening progress toward elimination.¹

National implications: NVBDCP and diagnostic procurement strategies

Although national prevalence of pfhrp2/3 deletions remains below the WHO 5% threshold in India, substantial heterogeneity has been documented in some states. This underscores the need for state specific surveillance rather than dependence on national averages. Under the NVBDCP and National Framework for Malaria Elimination (2016–2030), incorporating periodic molecular surveillance into routine monitoring can direct diagnostic decisions. By slowly introducing non-HRP2-based RDTs in selected hotspots, India’s efforts to eradicate malaria would be strengthened and diagnostic failure would be reduced.³⁹

Ultrasensitive HRP2-based RDTs

Ultrasensitive HRP2-based RDTs (uRDTs) have emerged as a corresponding tool to molecular surveillance. This acts by allowing detection of significantly lower HRP2 antigen concentrations than standard RDTs. Studies have shown that, in low-transmission and elimination settings uRDTs enhance the detection of asymptomatic and low-density infections. However, uRDTs cannot replace molecular surveillance in deletion-prone regions because they remain dependent on HRP2 antigen, they fail to detect parasites with complete pfhrp2 deletions. uRDTs can be used alongside periodic molecular monitoring to enhance routine case detection.⁴⁰

The relation between pfhrp2/3 deletions, related parasite fitness costs, and the antimalarial drug resistance, especially artemisinin in resistance, is an important factor to consider. According to gene-editing and in vitro studies, Plasmodium falciparum suffers measurable fitness costs because of pfhrp2 and pfhrp3 deletions. It was observed that single and double deletions reduce relative fitness by approximately 8.7% and 11.3%, respectively, when compared to unedited progenitor parasite. Genotypes deletion reduces effectiveness due to fitness costs similar to those of drug-resistance mutations in the absence of significant selection.⁴¹

Although pfhrp2/3 deletions mainly affect diagnostic by producing false-negative HRP-based RDT results, recent genomic surveillance has shown these gene deletions can occur with drug resistance dynamics, including partial artemisinin resistance, and diagnostic selection, especially in areas with high ACT use and low transmission. Monitoring drug-resistance markers along with pfhrp2/3 deletion surveillance is crucial for identifying pathways that could compromise diagnosis and therapy effectiveness.⁴⁰

Studies report geographic overlap between pfhrp2/3 deletions and emerging artemisinin resistance markers, particularly kelch13 variants, in East Africa. High rates of deletion prevalence have been observed along with increasing kelch13 mutations in regions of Ethiopia and Eritrea. Although no causal relation is established, these concomitant supports WHO recommendations for integrated genomic surveillance of diagnostic failure and drug-resistance markers.³⁸

In contrast, most of the Asian regions, including India and the Greater Mekong Subregion, continued to report low prevalence. However, some focal hotspots like Odisha have exhibited substantial HRP-RDT failure associated with deletions. These trends suggest prolonged dependence HRP-based RDTs use may favour parasites lacking HRP genes, to survive and spread over time. Therefore, it is crucial to emphasize the need for routine, timely molecular surveillance to guide diagnostic decisions.

The widespread and regional variation of pfhrp2/3 gene deletions pose a serious challenge to malaria diagnosis using HRP2-based RDTs. High deletion rates have been reported across various regions like Africa, Asia, and South America, leading to false negatives and thus affecting the control efforts. Continuous genomic surveillance, diagnostic modification, and alternate policy adaptation are important to maintain diagnostic accuracy. Alternatives to non-HRP2-based tests in high-deletion areas and adopting novel diagnostic tools are essential to ensuring effective malaria detection and supporting global elimination strategies.^42,43