Malaria, caused by various species of the Plasmodium parasite, remains a significant health threat in most U.S. military regions—AFRICOM, CENT­COM, INDOPACOM, and SOUTHCOM—and although less prevalent, also poses periodic risks to military personnel in NORTHCOM through imported cases. Early diagnosis is crucial for effective malaria chemotherapy, and rapid diagnostic tests have proven valuable in resource-poor settings and operational environments. The BinaxNow Malaria RDT is currently the sole U.S. Food and Drug Administration-approved test for use on U.S. military personnel. This simple RDT targets Plasmodium falciparum, the deadliest malaria species, by detecting the histidine-rich protein 2, as well as pan-Plasmodium species by detecting aldolase. The emergence of mutant P. falciparum parasites lacking pfhrp2/pfhrp3 genes and thus not expressing HRP2/HRP3 proteins poses a significant challenge in many malaria-endemic areas. This genetic variation has led to false-negative results in all HRP2-detecting RDTs including BinaxNow, undermining its utility. Current U.S. military force health protection measures for preventing malaria, including chemoprophylaxis, permethrin-treated uniforms, and DEET application to exposed skin, are effective, but breakthrough infections still occur. The use of portable and user-friendly malaria diagnostics is necessary in remote locations that lack microscopy or nucleic acid-based diagnostic capabilities. The alarmingly high prevalence of mutant pfhrp2/3-deleted parasites poses a threat to malaria diagnosis in all Combatant Commands where point-of-care testing is vital. This review emphasizes the importance of ongoing monitoring to determine the frequency and distribution of mutant parasites. Urgent attention is needed to develop alternative RDTs that can effectively detect malaria infections caused by these mutant strains.

What are the new findings?

These findings confirm that mutant pfhrp2/3-deleted parasites are highly prevalent in SOUTHCOM and parts of AFRICOM, rendering HRP2-based RDTs such as BinaxNow an unsuitable diagnostic tool for malaria in many of the SOUTHCOM and AFRICOM countries surveyed: Peru (14.3-62% between 2011-2018), Eritrea (62% in 2016 and 9.4% in 2020), Nigeria (13.3%), Sudan (11.2%), South Sudan (17.7%), and Uganda (3.3%). In INDOPACOM countries surveyed, no prevalence greater than 5% pfhrp2 deletions were observed. It is critical to continue surveillance on the frequency and distribution of these mutant parasites and develop alternative RDTs.

What is the impact on readiness and force health protection?

WHO recommends that countries switch to non-HRP2-based RDTs when prevalence of pfhrp2/3 deletions that cause false-negative RDT results exceed 5%. Current prevalence of mutant pfhrp2/3-deleted parasites causing false-negative RDT results has exceeded this threshold in most parts of SOUTHCOM and several areas of AFRICOM. If alternative diagnostic tests are not utilized in areas affected, life-saving malaria treatment for U.S. military personnel could be delayed. Continuous mapping of the frequency and distribution of mutant parasites directly informs FHP protection policy decisions for alternative diagnostic tool utilization.

Background

Malaria remains a major health problem worldwide. In 2021 the global burden of malaria was estimated at 247 million cases, an increase of 2 million cases from 2020.¹ In 2021, 20 malaria cases were reported among U.S. military personnel,² and this low number of reported cases, despite the massive global burden of malaria, is either an indication that current U.S. Department of Defense countermeasures (e.g., insecticide-treated uniforms, chemoprophylaxis drugs) are currently effective despite increasing mosquito resistance to permethrin and Plasmodium drug resistance, or because few U.S. military personnel are currently in areas of high malaria transmission. 

Malaria is caused by the Plasmodium apicomplexan protozoa parasite, a diverse genus of multiple hosts transmitted by a number of Anopheles genus mosquitoes. The Plasmodium species that commonly cause human malaria disease are Plasmodium falciparum, P. vivax, P. ovale, P. cynomolgi, and P. knowlesi. While each of these parasites can cause incapacitation unless appropriately treated, P. falciparum is rapidly fatal if not diagnosed and treated properly. Additionally, P. vivax, P. ovale, and P. cynomolgi have a dormant phase in the liver called hypnozoites that can relapse weeks to months later, causing debilitation and affecting readiness until properly cleared with the 8-aminoquinoline class of drugs. 

While microscopy is the gold standard for detecting Plasmodium parasites, it requires equipment and a competent microscopist not always available in austere environments. U.S. military operations often require movements far from fixed facilities with competent microscopists that can accurately diagnose malaria, and the weight of the diagnostic kit and its reliance on power and reagents is a significant consideration, which makes lightweight, easy-to-use RDTs more attractive than light microscopes. The World Health Organization recommends rapid diagnostic tests as a diagnostic tool when quality microscopy is not available.³ Despite many brands of malaria RDTs (89 meeting WHO procurement criteria and 11 WHO pre-qualified) that are commercially available, BinaxNOW Malaria RDT is the only RDT approved by the U.S. Food and Drug Administration for use in the U.S. and by U.S. military personnel, since 2007. 

BinaxNOW detects 2 Plasmodium-specific proteins: histidine-rich protein 2 (HRP2), specifically diagnosing P. falciparum, and aldolase, a pan-Plasmodium protein generically detecting all Plasmodium species. According to the WHO round 1 (2008) product testing of BinaxNOW, the detection rate for P. falciparum is 91.14% and P. vivax is 10% at 200/uL; for P. falciparum it is 100% and P. vivax 85% at 2000/uL.⁴ Performance characteristics of diagnostic tests can vary depending on several factors, including the expertise of the operator, the quality of the test kit, and the population tested. 

A serious threat to the utility of HRP2-based RDTs including BinaxNow has developed with the emergence of mutant P. falciparum parasites with deleted genes that encode HRP2 or a cross-reactive HRP3 which reduces or eliminates HRP2/3 protein expression, the targets of P. falciparum detection in BinaxNOW Malaria RDTs. These mutant parasites cause false-negative results. Pfhrp2/3-deleted P. falciparum parasites were first reported from patient samples collected between 2003 and 2007 in Peru,⁵ and have now been confirmed in 40 of 47 countries surveyed.¹ The overall pooled prevalence of pfhrp2/3-deleted parasites is highest in South America, followed by Africa, then Asia⁶; countries from South America and the Horn of Africa are among the worst-affected by pfhrp2/3-deleted mutant parasites. 

In 2019, WHO issued a “Response Plan to pfhrp2 Gene Deletions” outlining major strategies that include defining the frequency and distribution of mutant parasites, changing to non-HRP2-based RDTs when prevalence of pfhrp2/3 deletions that cause false-negative RDT results exceeds 5%, and developing new RDTs.⁷ Over the past 7 years, our team has collaborated with the WHO, national ministries of Health, and partner institutions to map and characterize pfhrp2/3-deleted parasites in several countries. These surveys provided critical data not only for national MoH diagnosis policy guidance, but also provided evidence of the extent of pfhrp2/3 deletions within U.S. Africa Command (AFRICOM), U.S. Indo-Pacific Command (INDOPACOM), and U.S. Southern Command (SOUTHCOM), to directly support Force Health Protection policy decisions. Herein we summarize major survey findings and their implications.

Methods

This is a review article collating and summarizing our survey findings published over the past seven years, and their implications for public health and FHP. Field surveys conducted by our collaborating teams are described in the Results; the laboratory methods used for determining pfhrp2/3 deletions are previously reported¹¹; 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 PCR¹¹; the work flow is summarized as a flow chart (Figure).

Results

AFRICOM AOR

Eritrea

Eritrea’s seasonal malaria transmission is low. Eritrea’s MoH introduced a HRP2-based combination RDT in 2006, but received complaints of false-negative RDT results in 2015. Initial investigations into possible causes of RDT failures led to suspicions about the presence of pfhrp2/3-deleted parasites. In 2016, the country’s MoH collected patient samples (n=50) from 2 hospitals in the Northern Red Sea Zone, where false-negative RDT results had been reported, to search for pfhrp2/3-deleted parasites. Laboratory analysis of these samples identified that 62% of patients were infected with pfhrp2/3-double-deleted parasites, and a further 20% with pfhrp3-deleted parasites. All double-pfhrp2/3-deleted parasites produced negative RDT results.⁸ Genetic relatedness analysis suggested that pfhrp2/3-deleted parasites in Eritrea likely emerged de novo. 

A follow-up survey in 2019 assessed the trend of pfhrp2/3-deleted parasites in Eritrea 2.5 years after the country’s MoH switched from HRP2-based RDTs. This was the first survey in the world to assess the epidemiology and evolution of mutant pfhrp2-deleted parasites following RDT switch. The survey collected P. falciparum samples from malaria patients at nine health facilities in three zones (n=715). Our analysis revealed an overall prevalence of mutant parasites lacking pfhrp2, pfhrp3, and both pfhrp2/3 genes of 9.4%, 41.7%, and 7.6%, respectively. The prevalence of mutant parasites was variable both within and between different zones.⁹ We also showed that the genetic diversity of mutant parasites significantly increased. While the prevalence of pfhrp2/3 deletions at all nine survey sites was lower than in 2016 at a different location, the overall prevalence of gene deletions in 2020 still exceeded the WHO threshold for RDT switch.  

Nigeria, Sudan and South Sudan

Because field surveys in many African countries are challenging, we analyzed imported P. falciparum cases (n=210, collected 2010-2018) to determine if pfhrp2/3-deleted parasites were present. We detected gene deletions in patients from 12 of 25 countries: pfhrp2-deletions in those from Nigeria (13.3%, n=30), Sudan (11.2%, n=39), and South Sudan (17.7%, n=17), and low levels of parasites with pfhrp3-deletion from Sudan (3.6%) and South Sudan (5.9%). No parasites with double-pfhrp2/3 deletions were detected.⁹ Microsatellite typing of parasites from Nigeria, Sudan, and South Sudan revealed low relatedness among gene-deleted parasites, indicating independent emergencies.

Uganda

Uganda has one of highest malaria burdens in Africa. In collaboration with the country’s MoH, we analyzed 300 P. falciparum isolates collected from cross-sectional malaria surveys in symptomatic individuals from 48 districts of Uganda’s Eastern and Western regions. The prevalence of parasites with pfhrp2, pfhrp3, and dual pfhrp2/3 deletions was 3.3%, 3.0%, and 3.3%, respectively.¹¹ The proportion of pfhrp2/3 deletions was higher in the Eastern (14.7%) compared to the Western region (3.1%). This is the first large-scale survey reporting the presence of pfhrp2/3-deleted parasites in Uganda. These mutant parasites contributed to 12.3% of false-negative RDT results, along with low parasite density and non-P. falciparum infections.¹² Genetic analyses showed a high rate of multiplicity of infections consistent with high transmission intensity in survey areas, and that gene-deleted parasites likely evolved de novo from the local parasite population.¹³ 

SOUTHCOM AOR

Peru has low malaria transmission, and despite the fact RDT is not a diagnostic mainstay for malaria in Peru, it was the first country to report pfhrp2/3-deleted parasites. In collaboration with Naval Medical Research Unit-6, we characterized the presence of pfhrp2 and pfhrp3 genes on P. falciparum samples (n=325) collected in Iquitos and surrounding communities between 2011 and 2018, as part of an ongoing project, Malaria Disease in Peru, to understand the prevalence trend of pfhrp2/3-deleted parasites and evolution over an 8-year period. Overall, double-pfhrp2 and -pfhrp3 deletions were detected in 67% of patient samples. We observed a concordance (Cohen’s Kappa=0.842) between pfhrp2 gene deletion and negligible HRP2 protein levels. Prevalence of gene deletion varied by study site, but the overall prevalence increased between 2011 (14.3%) and 2016 (88.4%), stabilizing around 65% in 2018.¹⁴ This prevalence increase was associated with rapid expansion of a single new parasite haplotype with double-pfhrp2/3 deletions. Our study showed the increase of pfhrp2/3 deletions in the absence of RDT pressure resulted from a clonal replacement of circulating lines with gene-deleted parasites in the Peruvian Amazon basin, suggesting that low immunity in the community to the new strain is likely the major factor in the rapid spread of pfhrp2/3 deletion. Interestingly, participants infected with double-pfhrp2/3-deleted parasites had a significantly lower parasitemia than those without gene deletions, which may cause less disease. 

INDOPACOM AOR

Pfhrp2-deleted parasites have been reported in India,¹⁵ the China-Myanmar border,¹⁶ and Indonesia,¹⁷ with variable prevalence of 0-25%, 4% and 4%, respectively. To date, there are no published survey reports of pfhrp2-deleted mutant parasites from other countries in this region.

Discussion

The rise of pfhrp2/3-deleted parasites is of significant concern for FHP, with current U.S. military personnel reliance on BinaxNOW malaria RDTs as the point-of-care test in austere environments. Deployed small teams of military personnel are most at risk of developing malaria and are simultaneously most reliant on RDTs. The gold standard for malaria diagnosis is by trained microscopist via blood smear. Lack of trained microscopists, however, has led many countries to become reliant upon malaria RDTs for diagnosis. In semi-fixed facilities with sufficient power and specialized equipment, nucleic acid-based methods are available for Plasmodium diagnosis that circumvents the HRP deletion issue. For deployed forces, this diagnostic capability such as a Biofire, Genexpert, or Loop Mediated Isothermal Amplification testing can be utilized at a Role 2 field hospital.  

P. falciparum is biodiverse among strains, so stratifying risk based on location is complex and requires active surveillance. These data presented summarize data collected from collaborating countries since 2016. There are inherent biases when focusing on a subset of all data collected on a topic, but our data concur with other published work on HRP2/3 deletions. Our DOD Global Emerging Infections Surveillance-supported findings contributed to the global effort of mapping the frequency of gene-deleted parasites causing false-negative RDT results and inform diagnostic policies of related stakeholders. Our 2016 survey in Eritrea was the first study demonstrating a high prevalence of gene-deleted parasites causing a high rate of RDT failures in Africa, and based on these survey findings, Eritrea switched to non-HRP2-based RDTs in 2016. The results from a 2019 follow-up survey suggest that HRP2-based RDT use is likely the main factor behind the high prevalence of gene-deleted parasites. Switching to non-HRP2-based RDT was partially effective in reducing gene-deleted parasite prevalence in Eritrea. HRP2-based RDTs remain unsuitable for malaria diagnosis in Eritrea at this time, however. 

We detected significant proportions of mutant parasites in travelers returning from Nigeria, Sudan, and South Sudan, where surveys are difficult to conduct. The proportions of parasites with gene deletions in those returning travelers signify a risk of false-negative HRP2 RDT results. In many of these countries our findings provided the first report of mutant parasites, which warrants surveillance to determine whether the prevalence of gene-deleted parasites justifies switching malaria RDTs in Nigeria, Sudan, and South Sudan.  

Our detection of gene-deleted parasites in Eastern and Western Uganda led to a nationwide survey to establish the prevalence and distribution of these mutant parasites. Field collection is complete and genetic analysis of samples is underway. We are also conducting surveillance of pfhrp2/3-deleted parasites in Cambodia and Papua New Guinea (PNG) in collaborations with the Armed Forces Research Institute of Medical Sciences and the PNG Defence Force.

Our microsatellite genotyping and genetic relatedness analyses revealed that pfhrp2/3-deleted parasites likely evolved de novo from local parasite populations. This finding suggests that a control rather than containment strategy may be a more effective intervention. These mutant parasites are prevalent in most malaria-endemic areas surveyed, with the ability to develop de novo, making false-negative malaria diagnosis a concern for all COCOMS. 

Forshey et al. provide a framework for continued assessment of the pfhrp2/3 deletion problem and a framework for U.S. DOD to acquire alternative RDTs that meet DOD requirements including FDA clearance.¹⁸ Currently, there are limited alternative RDTs available for the detection of parasites lacking HRP2. The WHO product testing round 8 evaluated 34 brands of RDTs against pfhrp2/3-deleted parasites and revealed that only two RDTs targeting Pan-pLDH met WHO procurement criteria, while RDTs specifically targeting Pf-pLDH performed poorly against gene-deleted parasites.¹⁹ While the two Pan-pLDH RDTs can be used to detect gene-deleted parasites in areas where P. falciparum is dominant, they are not WHO pre-qualified, nor FDA approved. For areas where discriminating P. falciparum from P. vivax and other Plasmodium species is necessary, combination RDTs detecting Pf-pLDH/Pv-pLDH and Pf-pLDH/pan-pLDH are required; a new Pf-pLDH RDT has shown promising performance against gene-deleted parasites,²⁰ and some are in the WHO pre-qualification pipeline. 

With the U.S. military operational framework evolving from counterinsurgency to large scale combat operations, doctrine is shifting to a need for prolonged casualty care that would benefit from a next-generation malaria RDT. For FHP in the short- and medium terms, FHP officers and pre-deployment planners should be made aware of the problem of pfhrp2/3-deleted parasites and given the most up-to-date aggregate surveillance data for their areas of responsibility. Long-term investment should be made in point-of-care testing with alternative targets that can discriminate different Plasmodium species and detect mutant parasites lacking HRP2.

Author Affiliations

Walter Reed Army Institute of Research Engineering and Scientist Exchange Program, Enoggera, QLD, Australia: MAJ Vesely (MSC, USA); Australian Defence Force Malaria and Infectious Disease Insti­tute, Enoggera, QLD, Australia: Dr. Cheng.

Acknowledgements

The authors thank our many collaborators and partner institutions in the publications referenced, and GEIS for continued support of sample analysis under P0160_23_AI. 

Disclaimer

This material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation or publica­tion. The opinions and assertions contained herein are the private views of the author, and are not to be construed as official, nor as reflecting true views of the Department of the Army or the Department of Defense.

The opinions expressed are those of the authors and do not necessarily reflect those of the Australian Defence Force.

References

1. World Health Organization. World Malaria Report 2022. World Health Organization; 2015. Accessed Apr. 3, 2023. https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2022
2. Armed Forces Health Surveillance Division. Update: malaria, U.S. Armed Forces, 2021. MSMR. 2022;29(3):2-7.
3. WHO. Guidelines for the Treatment of Malaria, 3rd edition. World Health Organization; 2015. Accessed Apr. 3, 2023. https://www.afro.who.int/publications/guidelines-treatment-malaria-third-edition
4. UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, Centers for Disease Control (U.S.), and Foundation for Innovative New Diagnostics. Malaria Rapid Diagnostic Test Performance--Results of WHO Product Testing of Malaria RDTs: Round 1 (2008). 2009. Accessed Apr. 3, 2023. https://apps.who.int/iris/handle/10665/44120
5. Gamboa D, Ho MF, Bendezu J, et al. A large proportion of P. falciparum isolates in the Amazon region of Peru lack pfhrp2 and pfhrp3: implications for malaria rapid diagnostic tests. PLoS One. 2010;5(1):e8091.
6. Jejaw Zeleke A, Hailu A, Bayih AG, et al. Plasmodium falciparum histidine-rich protein 2 and 3 genes deletion in global settings (2010-2021): a systematic review and meta-analysis. Malar J. 2022;21(1):26.
7. WHO. Response Plan to pfhrp2 Gene Deletions. 2019. Accessed Apr. 3, 2023. https://www.who.int/publications/i/item/WHO-CDS-GMP-2019.02
8. Berhane A, Anderson K, Mihreteab S, et al. Major threat to malaria control programs by Plasmodium falciparum lacking histidine-rich protein 2, Eritrea. Emerg Infect Dis. 2018;24(3):462-470.
9. Mihreteab S, Anderson K, Pasay C, et al. Epidemiology of mutant Plasmodium falciparum parasites lacking histidine-rich protein 2/3 genes in Eritrea 2 years after switching from HRP2-based RDTs. Sci Rep. 2021;11(1):21082.
10. Prosser C, Gresty K, Ellis J, et al. Plasmodium falciparum histidine-rich protein 2 and 3 gene deletions in strains from Nigeria, Sudan, and South Sudan. Emerg Infect Dis. 2021;27(2):471-479.
11. Bosco AB, Anderson K, Gresty K, et al. Molecular surveillance reveals the presence of pfhrp2 and pfhrp3 gene deletions in Plasmodium falciparum parasite populations in Uganda, 2017-2019. Malar J. 2020;19(1):300.
12. Bosco AB, Nankabirwa JI, Yeka A, et al. Limitations of rapid diagnostic tests in malaria surveys in areas with varied transmission intensity in Uganda 2017-2019: implications for selection and use of HRP2 RDTs. PLoS One. 2020;15(12):e0244457.
13. Bosco AB, Anderson K, Gresty K, et al. Genetic diversity and genetic relatedness in Plasmodium falciparum parasite population in individuals with uncomplicated malaria based on microsatellite typing in Eastern and Western regions of Uganda, 2019-2020. Malar J. 2021;20(1):242.
14. Valdivia HO, Anderson K, Smith D, et al. Spatiotemporal dynamics of Plasmodium falciparum histidine-rich protein 2 and 3 deletions in Peru. Sci Rep. 2022;12(1):19845.
15. Bharti PK, Chandel HS, Ahmad A, Krishna S, Udhayakumar V, Singh N. Prevalence of pfhrp2 and/or pfhrp3 gene deletion in Plasmodium falciparum population in eight highly endemic states in India. PLoS One. 2016;11(8):e0157949.
16. Li P, Xing H, Zhao Z, et al. Genetic diversity of Plasmodium falciparum histidine-rich protein 2 in the China-Myanmar border area. Acta Trop. 2015;152:26-31.
17. MalariaGen, Ahouidi A, Ali M, et al. An open dataset of Plasmodium falciparum genome variation in 7,000 worldwide samples. Wellcome Open Res. 2021;6:42. Accessed Apr. 3, 2023. https://www.who.int/teams/global-malaria-programme/reports/world-malaria-report-2022
18. Forshey BM, Morton L, Martin N, et al. Plasmodium falciparum rapid test failures threaten diagnosis and treatment of U.S. military personnel. Mil Med. 2020;185(1-2):e1-e4. doi:10.1093/milmed/usz256Fo
19. WHO. Malaria Rapid Diagnostic Test Performance—Results of WHO Product Testing of Malaria RDTs: Round 8 (2016-2018). 2018. Accessed Apr. 3, 2023. https://www.who.int/publications/i/item/9789241514965
20. Niyukuri D, Sinzinkayo D, Troth EV, et al. Performance of highly sensitive and conventional rapid diagnostic tests for clinical and subclinical Plasmodium falciparum infections, and hrp2/3 deletion status in Burundi. PLOS Glob Public Health. 2022;28(2[7]):e0000828. doi: 10.1371/journal.pgph.0000828