First detection of pfhrp2 and pfhrp3 gene deletions in Niger Republic: a retrospective sub- analysis of biological samples

First detection of pfhrp2 and pfhrp3 gene deletions in Niger Republic: a retrospective sub- analysis of biological samples | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article First detection of pfhrp2 and pfhrp3 gene deletions in Niger Republic: a retrospective sub- analysis of biological samples Ibrahima ISSA, Illa HACHIMOU, Djiby SOW, Lamine MAHAMAN MOUSTAPHA, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9407357/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background Rapid diagnostic tests (RDTs) based on histidine-rich protein 2 (HRP2) are the main diagnostic tool for malaria in Niger and many countries in sub-Saharan Africa. However, deletions of the P. falciparum hrp2 and hrp3 genes can compromise RDT performance, and pose a threat to diagnostic accuracy. Although these deletions were reported in several regions of Africa, Asia and South America, no molecular data on pfhrp2/3 were previously available for Niger. Methods The present study is a retrospective sub-analysis of biological samples derived from a therapeutic efficacy study (TES) conducted in Niger in 2022. Antigen profiling was performed using a multiplex bead-based assay to identify samples with weak or undetectable HRP2 signal. Species confirmation was conducted by PET-PCR, and pfhrp2 /3 exon 2 genotyping was performed using one-step PCR. Results Among the 375 P. falciparum mono-infection isolates analyzed, Pfhrp2 deletions were found in 11/375 (3.0%), Pfhrp3 deletions in 41/375 (10.9%), and double deletions in 5/375 (1.3%) samples. The highest prevalence of site-specific Pfhrp2 deletions (5.0%; 4/80) was observed at Baban Tabki (Zinder). Conclusions This is the first molecular evidence of Pfhrp2 and Pfhrp3 deletions in P in Niger. The Pfhrp2 deletion rate remains below the 5% threshold set by WHO for the revision of the RDT policy. It’s important to use WHO protocole for Pfhrp2 /3 deletion to determine the national prevalence of these genes deletion in Niger. P. falciparum Pfhrp2/3 HRP2-based RDTs gene deletion Malaria diagnostic Niger Figures Figure 1 Figure 2 Introduction Malaria remains a major public health problem in sub-Saharan Africa, where Plasmodium falciparum ( P. falciparum ) is responsible for the majority of cases and death. Since the 2000s [ 1 ]. In 2024, Niger accounted for approximately 6.1% of all malaria deaths in the WHO African Region, ranking 3rd among the countries most affected by the disease [ 2 ]. The main strategy for malaria control relies on rapid and accurate diagnosis, followed by effective treatment. Early and accurate diagnosis is essential for both effective disease management and adequate malaria surveillance. The quality of malaria diagnosis is important in all settings, as misdiagnosis can lead to significant morbidity and mortality. In Niger, other Plasmodium species also circulate, including Plasmodium vivax , Plasmodium ovale , and Plasmodium malariae [ 3 ]. The involvement of these species in malaria in Niger is significant because they can cause relapses, particularly Plasmodium vivax and Plasmodium ovale , and complicate diagnosis and treatment due to drug resistance. Global control efforts have led to a significant reduction in morbidity and mortality, largely due to the widespread use of key measures such as insecticide-treated nets [ 4 ], indoor spraying, Artemisinin-based Combination Therapies (ACTs) [ 5 ] and rapid diagnostic tests (RDTs). Among these tools, RDTs based on the histidine-rich protein 2 (HRP2), exclusively found in P. falciparum , have become the preferred method for rapidly diagnosing P. falciparum , especially in rural areas with limited resources to perform malaria microscopy or PCR. The ease of use, moderate cost, improved performance over pLDH RDTs, and independent product testing of HRP2-based RDTs explain their widespread adoption, particularly in Niger, where more than 90% of diagnoses are carried out with this type of test [ 6 ]. Since 2008, Niger has introduced the use of RDTs for malaria [ 7 ]. This initiative aims to improve the management of malaria cases, particularly in remote areas where access to microscopy services is limited. However, the reliability of HRP2-based RDTs is threatened by the emergence of deletions of pfhrp2 and pfhrp3 in P. falciparum , the genes encoding the HRP2 and/or HRP3 proteins decected by HRP2-based RDTs which can lead to false-negative results. Initially described in 2010 in the Amazon region of Peru [ 8 ], these deletions have since been reported in several regions of Africa [ 9 , 10 ], Asia [ 11 ] and South America [ 12 ]. The prevalence of parasites carrying Pfhrp2/3 deletions varies considerably from country to country, from less than 1% to more than 60%. In some areas, such as Eritrea, Djibouti or South Sudan, deletion rates exceed 5% [ 13 ]. However, the WHO has currently not set a threshold for levels of pfhrp2/3 genes within the parasite population, instead WHO recommend that countries should consider shifting away from exclusive reliance on HRP-based tests, and move instead to dual-antigen tests, pLDH-based tests, or microscopy when ≥ 5% of P. falciparum infections are missed by HRP2-based tests due to gene deletions (i.e. gene deletiosn cause ≥ 5% of HRP2-based RDTs produce false-negative test results). In the absence of available national data, the situation of pfhrp2/3 gene deletion in Niger remained unknown until now. Given the dependence of the Nigerien health system on HRP2 RDTs for malaria diagnosis, it became urgent to assess the potential presence of parasites carrying deletions of the Pfhrp2 and Pfhrp3 genes. The present study aims to determine the circulation of P. falciparum strains carrying hrp2/hrp3 deletion in Niger, using samples from a multicenter therapeutic efficacy study of artemeter lumefantrine. Methods Study design The present study is a retrospective sub-analysis of biological samples derived from a therapeutic efficacy study (TES) conducted in Niger in 2022 [ 14 ]. The methodology of the primary study, which focused on the recruitment of patients with uncomplicated P. falciparum malaria, has been previously described in detail [ 14 ]. For the current genomic surveillance of hrp2 and hrp3 genes deletions, inclusion was restricted to Day 0 (pre-treatment) samples. All dried blood spots (DBS) with thick smear positive collected at Day 0 were initially screened using Enzyme-Linked Immunosorbent Assay (ELISA). Subsequently, samples confirmed as P. falciparum mono-infections underwent molecular genotyping to detect the presence or deletion of the pfhrp2 and pfhrp3 genes. Multiplex antigen detection Antigenic evaluation was performed using the Luminex MAGPIX platform, according to the protocols of Rogier E. et al [ 15 ], for the simultaneous detection of the following antigens: pHRP2, pAldolase (pan-plasmodial aldolase), pLDH (pan-specific lactate dehydrogenase), pfLDH ( P. falciparum ), PvLDH ( P. vivax ) and poLDH ( P. ovale ). The antigenic profiles were visualized as a scatterplot plots, as described previously [ 16 ]. Samples with an absent or significantly below threshold HRP2 signal and one or more other elevated antigens were considered suspect of pfhrp2 gene deletion and selected for molecular analysis. DNA extraction The extraction of the parasitic DNA was carried out using the QIAamp® DNA Mini (Qiagen) kit, according to the manufacturer's recommendations. The protocol had two main steps: cell lysis and DNA purification. The latter included enzymatic digestion by proteinase K, followed by two successive washes using AW1 and AW2 buffers allowing the removal of proteins, contaminants, enzymes and PCR inhibitors. The final elution of the DNA was performed with 80 µL the AEbuffer. The DNA extracts were then stored at -20°C until they were used. Quality control of P. falciparum by genotyping of the msp1 and msp2 genes To verify the quality and integrity of the extracted DNA, as well as to specifically confirm the presence of P. falciparum , we performed PCR amplification targeting two monotypic genes encoding merozoite surface proteins Pfmsp1 (merozoite surface protein 1) and Pfmsp2 (merozoite surface protein 2). These markers, strictly present in a single copy per parasite in the genome, offer a rigorous control of DNA amplification [ 17 ]. The primers used for Pfmsp1 and Pfmsp2 (Supplementary table 1 ), described in S. Viriyakosol et al.[ 17 ] and Felger et al. [ 18 ], made it possible to obtain fragments of expected size via a two-step protocol, as detailed in the WHO manual [ 19 ]. Species confirmation The samples were analyzed by multiplex real-time PCR (PET-PCR or photo-inducted electron transfer) in order to identify the Plasmodium species [ 20 ]. Only confirmed monospecific P. falciparum infections were retained for subsequent analyses. PET-PCR allows both the confirmation of infection and the quantification of the genome at the level of the genus Plasmodium as well as the species P. falciparum . In summary, amplification targeting the genus Plasmodium was performed in a total reaction volume of 20 µL containing: 2X of ABI TaqMan® environmental buffer, 250 nM of each sense and antisense primer (with the exception of the specific primer of P. falciparum , labelled HEX, used at a concentration of 125 nM, see supplementary table 2. For each sample, two PET-PCR replicates were performed from 2 µL of template DNA. The thermocycling protocol included an initial activation at 95°C for 15 minutes, followed by 45 cycles including denaturation at 95°C for 20 seconds, then hybridization at 60°C for 40 seconds. A cycle threshold (Ct) of 35 was retained as the limit of positivity. Detection of deletions of the Pfhrp2 and Pfhrp3 genes Genotyping of the Pfhrp2 and Pfhrp3 genes was performed by PCR uniplex (one-step), targeting exon 2 of each of the two genes. Published primers known as "BRAVO" (Supplementary table 3) were used, with optimized amplification conditions [ 21 ]. The presence of a deletion was concluded when no amplification product of the targeted gene was observed despite the successful completion of internal controls (amplification of the second Pfhrp gene or PET-PCR confirmation), thus excluding a technical failure. Statistical analysis The antigen detection data was processed and visualized in R (version 3.6.1) using ggplot2. The positivity threshold was determined for each antigen by the log-normal mean plus three standard deviations from a panel of 86 negative samples. Samples exhibiting an absent or significantly lower HRP2 signal and one or more other elevated antigens were grouped together as suspected of pfhrp2 gene deletion and selected for molecular analysis. Results Antigen screening A total of 438 DBS were analyzed by Luminex MAGPIX multiplex. The antigenic profiles identified samples suspected for Pfhrp2 deletion. Figure 1 shows the distribution of the different antigens compared with detection of the Pfhrp2 antigen. Confirmation of P. falciparum infections by PET-PCR Among the 438 patients included, 375 mono-infection of P . falciparum infections were confirmed by PET-PCR (Table 1 ). The remaining 63 samples were excluded, due to detected co-infections ( P. malariae , P. ovale ) or discrepancies between antigenic and parasitological results. Table 1 Detection of Plasmodial infections Plasmodial species n % P. falciparum 375 85.6 P. falciparum + P. malariae 30 6.8 P. falciparum + P. ovale 20 4.6 P. falciparum + P. ovale + P. malariae 13 3.0 Total 438 100 Deletion of the Pfhrp2 and Pfhrp3 genes Among the 375 mono infection of P. falciparum isolate, 2.9% (11/375) had a deletion of the pfhrp2 gene , 10.9% (41/375) had a deletion of the pfhrp3 gene, and 1.3% (5/375) had a combined deletion of both genes. Table 2 below summarizes the spatial distribution of these deletions. The frequencies of pfhrp2 deletions ranged from 1.7% to 5.0% by site. Baban Tabki (Zinder) hab the high prevalence of pfhrp2 . The Pfhrp3 and double deletions Pfhrp2 / Pfhrp3 were found in all the study sites (Table 2 ). Table 2 Regional distribution of pfhrp2 and pfhrp3 deletions among P. falciparum infections (N = 375) Study site (Region) Samples pfhrp2 (%) pfhrp3 (%) pfhrp2/pfhrp3 (%) Aderbissinat (Agadez) 80 2 (2.5%) 12 (15.0%) 2 (2.5%) Aguié (Maradi) 100 3 (3.0%) 10 (10.0%) 1 (1.0%) Baban Tabki (Zinder) 80 4 (5.0%) 8 (10.0%) 1 (1.3%) Boboye (Dosso) 115 2 (1.7%) 11 (9.6%) 1 (0.9%) Total 375 11 (2.9%) 41 (10.9%) 5 (1.3%) Discussion Deletions of the pfhrp2 and pfhrp3 genes represent a growing threat to the diagnostic performance of HRP2-based rapid tests, widely used for the detection of P. falciparum in Africa. The present study identified the detection of these deletions in Niger. The analysis was carried out on samples derived from a therapeutic efficacy study (TES) conducted in Niger in 2022 [ 22 ]. Our study is the first to report the deletions of pfhrp2 and pfhrp3 in Niger, with a prevalence of 3% for pfhrp2 and 10.9% for pfhrp3 . The prevalence of pfhrp2 deletion remains below the 5%. The WHO recommends that countries consider switching away from exclusive reliance on HRP2-based tests when the rate of false-negative HRP2-based RDTs is greater or equal to 5% due to gene deletions [ 23 ]. It’s important to higligh that our study was not initialy disease as described by WHO to determine the prevalence of pfhrp2/pfhp3 deletion. Based on the identification of pfhrp2 and pfhrp3 deletions in Niger, further studies are required to determine the false negative rate of HRP2-based based on the WHO protocole. Furthermore, a geographical variation in deletion frequency was observed. The specific prevalence of the pfhrp2 deletion in Zinder is 5.0%. The heterogeneous distribution observed between the sites argues for distinct local dynamics. At Zinder, the high rate of pfhrp2-deletion could be related to proximity to northern Nigeria, where similar rates have been reported [ 24 ], suggesting a transmission of deleted parasites across national boundaries or shared selection pressure. In contrast, the situation in Agadez, a desertic region with low transmission, could reflect a founder effect or genetic drift in an isolated population [ 25 ]. In-depth genomic analysis (microsatellites, WGS sequencing) would provide a better understanding of the emergence and dispersion of these variants [ 25 ]. In contrast to our study, the highest prevalences were reported in Ethiopia (5.75% for pfhrp2 , 35.53% for pfhrp3 ) [ 26 ] and in some regions of East Africa [ 27 ], while several countries, including Togo [ 12 ] and Mozambique, reported no deletions [ 28 ]. Most West and Central Africa still reports sporadic or absent deletions [ 12 , 29 – 31 ] Regarding deletions of the Pfhrp3 gene, their high prevalence (up to 15% in Agadez) is part of a global trend observed in other African contexts, where losses of Pfhrp3 seem to occur more frequently than those of Pfhrp2 [ 32 ]. Parasites carrying combined deletions of both genes (1.3% in our study) are of particular concern, as they completely evade HRP2 RDTs, exposing patients to a risk of serious false negatives [ 33 ]. From a public health perspective, the continued use of HRP2-based RDTs remains justified at the national level. However, the presence of critical foci such as the one identified in Zinder calls for additional targeted investigations, particularly on symptomatic RDT-negative cases. If a prevalence of false-negative HRP2-based RDTs ≥ 5% is confirmed, the adoption of alternative diagnostic tests, such as dual-antigen HRP2/pLDH or RDTs targeting pan-plasmodial LDH or PfLDH antigens should be considered in circumstances where microscopy is not feasible, in line with WHO recommendations [ 34 ]. One limitation of this study was the use of samples which tested positive by both microscopy and HRP2-based RDT, potentially biasing the selection of samples to include wildtype pfhrp2/3 parasites. However, the fact that some Pfhrp2-negative parasites were still detected despite initial screening based on the HRP2 RDT suggests that the closely-related HRP3 protein may partially compensate for the absence of HRP2 due to the presence of similar repeat epitopes present in both proteins [ 35 ]. This phenomenon complicates the identification of deleted strains [ 36 ]. Conversely, it is also likely that some infections with parasites carrying Pfhrp2-deletion were wrongly excluded at the screening stage, due to a lack of RDT positivity [ 33 , 37 , 38 ]. This means that the true prevalence may be underestimated in our study. Another limitation is the antigen screening using multiplex bead-based assays used to prioritize samples for molecular genotyping; while this approach improves efficiency, it may fail to identify low-density infections or parasites with residual HRP2 or compensatory HRP3 expression, potentially leading to misclassification. Finally, although sentinel sites were selected to represent distinct epidemiological areas, the findings may not be fully generalizable to all regions of the country, particularly areas with different transmission intensities or diagnostic practices. Conclusion This study provides the first molecular data on the presence of deletions of the pfhrp2 and pfhrp3 genes in P. falciparum in Niger. The prevalence of pfhrp2 deletion remains below 5% among all samples. The higher frequency of pfhrp3 deletion and the identification of double-deletion strains reinforce the need for surveillance. Our findings support the establishment of an integrated molecular surveillance system, including genotyping of RDT-negative/microscopy positive cases, to anticipate diagnostic failures and adapt national malaria control policies. Abbreviations • ACTs Artemisinin-based Combination Therapies • AW1 / AW2 Wash buffers • bp Base pairs • CDC U.S. Centers for Disease Control and Prevention • CERMES Centre de Recherche Médical et Sanitaire (Niamey, Niger) • CIGASS International Research and Training Center in Applied Genomics and Health Surveillance (Dakar, Sénégal) • CNERS Comité National d’Éthique pour la Recherche en Santé (Niger) • Ct Cycle threshold • DBS Dried blood spots • DNA Deoxyribonucleic acid • ELISA Enzyme-Linked Immunosorbent Assay • HRP2 Histidine-rich protein 2 • HRP3 Histidine-rich protein 3 • MSP1 / Pfmsp1 Merozoite surface protein 1 • MSP2 / Pfmsp2 Merozoite surface protein 2 • pAldolase Pan-plasmodial aldolase • PCR Polymerase Chain Reaction (réaction de polymérisation en chaîne) • PET-PCR Photo-induced electron transfer - Polymerase Chain Reaction • pfhrp2 / pfhrp3 Gènes codant pour les protéines HRP2 et HRP3 chez P. falciparum • pfLDH P. falciparum lactate dehydrogenase • pLDH Pan-specific lactate dehydrogenase (lactate déshydrogénase commune aux espèces de Plasmodium ) • PMI U.S. President's Malaria Initiative • PNLP Programme National de Lutte contre Paludisme (Niger) • poLDH P. ovale lactate dehydrogenase • PvLDH P. vivax lactate dehydrogenase • RDTs (TDR) Rapid diagnostic tests • TES Therapeutic efficacy study • UAS Université André Salifou (Zinder, Niger) • USAID U.S. Agency for International Development • WGS Whole Genome Sequencing • WHO (OMS) World Health Organization Declarations Ethics approval and consent to participate: This study received authorization from the National Ethics Committee for Health Research (CNERS) on 12/07/2022 by letter referenced N°32/2022/CNERS. Consent for publication: Not applicable Availability of data and materials: All data generated or analysed during this study are included in this published article. Competing interests: The authors declare that there is no conflict of interest Funding: This study was funded by the U.S. President's Malaria Initiative Impact Malaria, Niger. Authors' contribution: I.M.L., I.I. and MKS: Contributed to the writing of the protocol, its implementation, the writing of the manuscript and the management of the teams. I.I and MML write the first and final draft of the manuscript. II and MKS: Participated in the collection, data analysis, molecular study in Senegal, and in the drafting of the report. A.B.D; M.A.D; D.S; J.M; I.C and AS: led the training and technical supervision of the researchers during the molecular analysis and also participated in the data analysis and writing of the manuscript. 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Malar J. 2020;19:392. https://doi.org/10.1186/s12936-020-03460-w . Baker J, Gatton ML, Peters J, Ho M-F, McCarthy JS, Cheng Q. Transcription and Expression of Plasmodium falciparum Histidine-Rich Proteins in Different Stages and Strains: Implications for Rapid Diagnostic Tests. PLoS ONE. 2011;6:e22593. https://doi.org/10.1371/journal.pone.0022593 . Gillet P, Mori M, Van den Ende J, Jacobs J. Buffer substitution in malaria rapid diagnostic tests causes false-positive results. Malar J. 2010;9:215. https://doi.org/10.1186/1475-2875-9-215 . Okai T, Chan CW, KC A, Omondi P, Musyoka K, Kongere J, et al. Plasmodium falciparum with pfhrp2 and pfhrp3 gene deletions in asymptomatic malaria infections in the Lake Victoria region, Kenya. Trop Med Health. 2024;52:94. https://doi.org/10.1186/s41182-024-00664-7 . Gyuricza IG, Fola AA, Simkin A, Thwai KL, Juliano JJ, Bailey JA et al. Malaria control and the unexpected spread of diagnostic-resistant Plasmodium falciparum in Peru [Internet]. bioRxiv; 2026 [cited 2026 Mar 11]. p. 2026.02.25.707493. https://doi.org/10.64898/2026.02.25.707493 . Additional Declarations No competing interests reported. Supplementary Files SupplementalMaterialsNigerHrp23.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 22 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 19 Apr, 2026 Reviewers invited by journal 17 Apr, 2026 Editor assigned by journal 15 Apr, 2026 Submission checks completed at journal 15 Apr, 2026 First submitted to journal 13 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9407357","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":627857493,"identity":"ac47f643-c24a-4301-a077-0049244a02c6","order_by":0,"name":"Ibrahima ISSA","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYBACNgkwJWHAwN7AwAxiAmlitfAcgGgB0gQARAuDAYNEApFa+KR7H36u+GNhzD/zjeHnggobBh5pAnrYZI4bS57hkTCTuJ1jLD3jTBoDD18CAS0SaQySDRISNgy3cwykedsOM9jzEHAYUAvzzwYDCRv5m2eMf4O08BChhU2yIUHCzOAGj5k0cVpkjrFZNhyQMDY8k1ZmzXMmjYegFvnZbcw3G/7UGc47fnjzbZ4KGzmCWpAAhwGIJEEDMKU8IEX1KBgFo2AUjCAAAEg/NQr/pQECAAAAAElFTkSuQmCC","orcid":"","institution":"Centre de Recherche Médicale et Sanitaire","correspondingAuthor":true,"prefix":"","firstName":"Ibrahima","middleName":"","lastName":"ISSA","suffix":""},{"id":627857503,"identity":"9ebbe549-61f5-48e9-b973-362f47f78b98","order_by":1,"name":"Illa HACHIMOU","email":"","orcid":"","institution":"Programme National de Lutte contre Paludisme (PNLP)","correspondingAuthor":false,"prefix":"","firstName":"Illa","middleName":"","lastName":"HACHIMOU","suffix":""},{"id":627857507,"identity":"1645fd0b-dab0-4fae-9eda-63c7bf9a2497","order_by":2,"name":"Djiby SOW","email":"","orcid":"","institution":"International Research and Training Center in Applied Genomics and Health Surveillance (CIGASS)","correspondingAuthor":false,"prefix":"","firstName":"Djiby","middleName":"","lastName":"SOW","suffix":""},{"id":627857510,"identity":"0b076c79-2244-434e-bee1-29ccd3059eb0","order_by":3,"name":"Lamine MAHAMAN MOUSTAPHA","email":"","orcid":"","institution":"Université André Salifou (UAS)","correspondingAuthor":false,"prefix":"","firstName":"Lamine","middleName":"MAHAMAN","lastName":"MOUSTAPHA","suffix":""},{"id":627857520,"identity":"cf73aa8c-da57-48d8-97fe-c6af843c0c15","order_by":4,"name":"Kabirou MAMANE SANOUSSI","email":"","orcid":"","institution":"Programme National de Lutte contre Paludisme (PNLP)","correspondingAuthor":false,"prefix":"","firstName":"Kabirou","middleName":"MAMANE","lastName":"SANOUSSI","suffix":""},{"id":627857522,"identity":"50a9fd30-0ed2-4f14-a301-4cba7f4f629b","order_by":5,"name":"Yacouba IDRISSA","email":"","orcid":"","institution":"Programme National de Lutte contre Paludisme (PNLP)","correspondingAuthor":false,"prefix":"","firstName":"Yacouba","middleName":"","lastName":"IDRISSA","suffix":""},{"id":627857527,"identity":"2960fb10-6c22-4b0f-a026-8076af37caa3","order_by":6,"name":"Mamadou ALPHA DIALLO","email":"","orcid":"","institution":"International Research and Training Center in Applied Genomics and Health Surveillance (CIGASS)","correspondingAuthor":false,"prefix":"","firstName":"Mamadou","middleName":"ALPHA","lastName":"DIALLO","suffix":""},{"id":627857530,"identity":"00a4a39a-b6eb-4292-88f5-0604d0845132","order_by":7,"name":"Aïta SENE","email":"","orcid":"","institution":"International Research and Training Center in Applied Genomics and Health Surveillance (CIGASS)","correspondingAuthor":false,"prefix":"","firstName":"Aïta","middleName":"","lastName":"SENE","suffix":""},{"id":627857532,"identity":"65182831-785f-4fd3-a67e-9fb8d1c062d4","order_by":8,"name":"Ibrahima MBAYE NDIAYE","email":"","orcid":"","institution":"International Research and Training Center in Applied Genomics and Health Surveillance (CIGASS)","correspondingAuthor":false,"prefix":"","firstName":"Ibrahima","middleName":"MBAYE","lastName":"NDIAYE","suffix":""},{"id":627857543,"identity":"c67e1c17-9ba8-4a22-bfc1-d3b8314d09e0","order_by":9,"name":"Zilahatou BAHARI-TOHON","email":"","orcid":"","institution":"U.S. President's Malaria Initiative, USAID","correspondingAuthor":false,"prefix":"","firstName":"Zilahatou","middleName":"","lastName":"BAHARI-TOHON","suffix":""},{"id":627857545,"identity":"c2df5564-d730-4e1a-870d-5e2af91477b9","order_by":10,"name":"Irene CAVROS","email":"","orcid":"","institution":"President's Malaria Initiative, U.S. Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Irene","middleName":"","lastName":"CAVROS","suffix":""},{"id":627857551,"identity":"56d29624-f18b-4b38-a4d3-0b5e966186e3","order_by":11,"name":"Jessica MCCAFFERY","email":"","orcid":"","institution":"President's Malaria Initiative, U.S. Centers for Disease Control and Prevention","correspondingAuthor":false,"prefix":"","firstName":"Jessica","middleName":"","lastName":"MCCAFFERY","suffix":""},{"id":627857552,"identity":"3c407343-142e-4e03-aad3-1d2b5d11c80a","order_by":12,"name":"Awa BINETA DEME","email":"","orcid":"","institution":"International Research and Training Center in Applied Genomics and Health Surveillance (CIGASS)","correspondingAuthor":false,"prefix":"","firstName":"Awa","middleName":"BINETA","lastName":"DEME","suffix":""},{"id":627857553,"identity":"588c6560-e4de-4711-a634-e177e27a2592","order_by":13,"name":"Laminou IBRAHIM MAMAN","email":"","orcid":"","institution":"Centre de Recherche Médicale et Sanitaire","correspondingAuthor":false,"prefix":"","firstName":"Laminou","middleName":"IBRAHIM","lastName":"MAMAN","suffix":""}],"badges":[],"createdAt":"2026-04-13 18:08:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9407357/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9407357/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107870363,"identity":"f48efb80-5547-4d5b-b0a5-6f6e01a89c25","added_by":"auto","created_at":"2026-04-27 07:39:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":315999,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of the different antigens according to the Pfhrp2antigen\u003c/strong\u003e Scatter plots of HRP2 antigen levels compared to those of other Plasmodium antigens. These plots represent data from 438 dried blood samples (DBS) analyzed by a Luminex MAGPIX assay for Plasmodium antigens. HRP2 antigen levels are compared to those of pfLDH, pAldolase, and pLDH. The red-yellow area represents DBS with low or absent HRP2 levels, selected for subsequent pfhrp2 genotyping due to a discordant antigenic signal between HRP2 and pan-Plasmodium aldolase or LDH antigens, or P. falciparum-specific LDH.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9407357/v1/e19b87cad9c542bfd6e3f77d.png"},{"id":107838510,"identity":"4affdeec-59f2-4544-8582-0f9f312707ce","added_by":"auto","created_at":"2026-04-26 17:10:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89301,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistribution of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epfhrp2\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003epfhrp3\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene deletion profiles across study site.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9407357/v1/755c50973ecbce296d587e4d.png"},{"id":108180988,"identity":"7507c886-82fd-46d5-a835-64a699e65479","added_by":"auto","created_at":"2026-04-30 08:55:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":657474,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9407357/v1/8584a8fe-d810-467c-b674-3af429c75d96.pdf"},{"id":107838508,"identity":"eb33e5a9-3b1e-4600-89a9-01c294da23aa","added_by":"auto","created_at":"2026-04-26 17:10:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":211958,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterialsNigerHrp23.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9407357/v1/91d16c7daebb87a7c191a0bd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"First detection of pfhrp2 and pfhrp3 gene deletions in Niger Republic: a retrospective sub- analysis of biological samples","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMalaria remains a major public health problem in sub-Saharan Africa, where \u003cem\u003ePlasmodium falciparum\u003c/em\u003e (\u003cem\u003eP. falciparum\u003c/em\u003e) is responsible for the majority of cases and death. Since the 2000s [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In 2024, Niger accounted for approximately 6.1% of all malaria deaths in the WHO African Region, ranking 3rd among the countries most affected by the disease [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The main strategy for malaria control relies on rapid and accurate diagnosis, followed by effective treatment. Early and accurate diagnosis is essential for both effective disease management and adequate malaria surveillance. The quality of malaria diagnosis is important in all settings, as misdiagnosis can lead to significant morbidity and mortality. In Niger, other \u003cem\u003ePlasmodium\u003c/em\u003e species also circulate, including \u003cem\u003ePlasmodium vivax\u003c/em\u003e, \u003cem\u003ePlasmodium ovale\u003c/em\u003e, and \u003cem\u003ePlasmodium malariae\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The involvement of these species in malaria in Niger is significant because they can cause relapses, particularly \u003cem\u003ePlasmodium vivax\u003c/em\u003e and \u003cem\u003ePlasmodium ovale\u003c/em\u003e, and complicate diagnosis and treatment due to drug resistance. Global control efforts have led to a significant reduction in morbidity and mortality, largely due to the widespread use of key measures such as insecticide-treated nets [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], indoor spraying, Artemisinin-based Combination Therapies (ACTs) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and rapid diagnostic tests (RDTs). Among these tools, RDTs based on the histidine-rich protein 2 (HRP2), exclusively found in \u003cem\u003eP. falciparum\u003c/em\u003e, have become the preferred method for rapidly diagnosing \u003cem\u003eP. falciparum\u003c/em\u003e, especially in rural areas with limited resources to perform malaria microscopy or PCR. The ease of use, moderate cost, improved performance over pLDH RDTs, and independent product testing of HRP2-based RDTs explain their widespread adoption, particularly in Niger, where more than 90% of diagnoses are carried out with this type of test [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Since 2008, Niger has introduced the use of RDTs for malaria [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This initiative aims to improve the management of malaria cases, particularly in remote areas where access to microscopy services is limited.\u003c/p\u003e \u003cp\u003eHowever, the reliability of HRP2-based RDTs is threatened by the emergence of deletions of \u003cem\u003epfhrp2\u003c/em\u003e and \u003cem\u003epfhrp3\u003c/em\u003e in \u003cem\u003eP. falciparum\u003c/em\u003e, the genes encoding the HRP2 and/or HRP3 proteins decected by HRP2-based RDTs which can lead to false-negative results. Initially described in 2010 in the Amazon region of Peru [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], these deletions have since been reported in several regions of Africa [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], Asia [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and South America [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The prevalence of parasites carrying \u003cem\u003ePfhrp2/3\u003c/em\u003e deletions varies considerably from country to country, from less than 1% to more than 60%. In some areas, such as Eritrea, Djibouti or South Sudan, deletion rates exceed 5% [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the WHO has currently not set a threshold for levels of \u003cem\u003epfhrp2/3\u003c/em\u003e genes within the parasite population, instead WHO recommend that countries should consider shifting away from exclusive reliance on HRP-based tests, and move instead to dual-antigen tests, pLDH-based tests, or microscopy when \u0026ge;\u0026thinsp;5% of \u003cem\u003eP. falciparum\u003c/em\u003e infections are missed by HRP2-based tests due to gene deletions (i.e. gene deletiosn cause\u0026thinsp;\u0026ge;\u0026thinsp;5% of HRP2-based RDTs produce false-negative test results).\u003c/p\u003e \u003cp\u003eIn the absence of available national data, the situation of \u003cem\u003epfhrp2/3\u003c/em\u003e gene deletion in Niger remained unknown until now. Given the dependence of the Nigerien health system on HRP2 RDTs for malaria diagnosis, it became urgent to assess the potential presence of parasites carrying deletions of the \u003cem\u003ePfhrp2\u003c/em\u003e and \u003cem\u003ePfhrp3\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003eThe present study aims to determine the circulation of \u003cem\u003eP. falciparum\u003c/em\u003e strains carrying \u003cem\u003ehrp2/hrp3\u003c/em\u003e deletion in Niger, using samples from a multicenter therapeutic efficacy study of artemeter lumefantrine.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design\u003c/h2\u003e \u003cp\u003eThe present study is a retrospective sub-analysis of biological samples derived from a therapeutic efficacy study (TES) conducted in Niger in 2022 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The methodology of the primary study, which focused on the recruitment of patients with uncomplicated \u003cem\u003eP. falciparum\u003c/em\u003e malaria, has been previously described in detail [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For the current genomic surveillance of \u003cem\u003ehrp2\u003c/em\u003e and \u003cem\u003ehrp3\u003c/em\u003e genes deletions, inclusion was restricted to Day 0 (pre-treatment) samples.\u003c/p\u003e \u003cp\u003eAll dried blood spots (DBS) with thick smear positive collected at Day 0 were initially screened using Enzyme-Linked Immunosorbent Assay (ELISA). Subsequently, samples confirmed as \u003cem\u003eP. falciparum\u003c/em\u003e mono-infections underwent molecular genotyping to detect the presence or deletion of the \u003cem\u003epfhrp2\u003c/em\u003e and \u003cem\u003epfhrp3\u003c/em\u003e genes.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMultiplex antigen detection\u003c/h3\u003e\n\u003cp\u003eAntigenic evaluation was performed using the Luminex MAGPIX platform, according to the protocols of Rogier E. et al [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], for the simultaneous detection of the following antigens: pHRP2, pAldolase (pan-plasmodial aldolase), pLDH (pan-specific lactate dehydrogenase), pfLDH (\u003cem\u003eP. falciparum\u003c/em\u003e), PvLDH (\u003cem\u003eP. vivax\u003c/em\u003e) and poLDH (\u003cem\u003eP. ovale\u003c/em\u003e). The antigenic profiles were visualized as a scatterplot plots, as described previously [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Samples with an absent or significantly below threshold HRP2 signal and one or more other elevated antigens were considered suspect of \u003cem\u003epfhrp2\u003c/em\u003e gene deletion and selected for molecular analysis.\u003c/p\u003e\n\u003ch3\u003eDNA extraction\u003c/h3\u003e\n\u003cp\u003eThe extraction of the parasitic DNA was carried out using the QIAamp\u0026reg; DNA Mini (Qiagen) kit, according to the manufacturer's recommendations. The protocol had two main steps: cell lysis and DNA purification. The latter included enzymatic digestion by proteinase K, followed by two successive washes using AW1 and AW2 buffers allowing the removal of proteins, contaminants, enzymes and PCR inhibitors. The final elution of the DNA was performed with 80 \u0026micro;L the AEbuffer. The DNA extracts were then stored at -20\u0026deg;C until they were used.\u003c/p\u003e \u003cp\u003e \u003cb\u003eQuality control of\u003c/b\u003e \u003cb\u003eP. falciparum\u003c/b\u003e \u003cb\u003eby genotyping of the\u003c/b\u003e \u003cb\u003emsp1\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003emsp2\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo verify the quality and integrity of the extracted DNA, as well as to specifically confirm the presence of \u003cem\u003eP. falciparum\u003c/em\u003e, we performed PCR amplification targeting two monotypic genes encoding merozoite surface proteins \u003cem\u003ePfmsp1\u003c/em\u003e (merozoite surface protein 1) and \u003cem\u003ePfmsp2\u003c/em\u003e (merozoite surface protein 2). These markers, strictly present in a single copy per parasite in the genome, offer a rigorous control of DNA amplification [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The primers used for \u003cem\u003ePfmsp1\u003c/em\u003e and \u003cem\u003ePfmsp2\u003c/em\u003e (Supplementary table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), described in S. Viriyakosol et al.[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and Felger \u003cem\u003eet al.\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], made it possible to obtain fragments of expected size via a two-step protocol, as detailed in the WHO manual [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eSpecies confirmation\u003c/h3\u003e\n\u003cp\u003eThe samples were analyzed by multiplex real-time PCR (PET-PCR or photo-inducted electron transfer) in order to identify the \u003cem\u003ePlasmodium\u003c/em\u003e species [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Only confirmed monospecific \u003cem\u003eP. falciparum\u003c/em\u003e infections were retained for subsequent analyses. PET-PCR allows both the confirmation of infection and the quantification of the genome at the level of the genus \u003cem\u003ePlasmodium\u003c/em\u003e as well as the species \u003cem\u003eP. falciparum\u003c/em\u003e. In summary, amplification targeting the \u003cem\u003egenus Plasmodium\u003c/em\u003e was performed in a total reaction volume of 20 \u0026micro;L containing: 2X of ABI TaqMan\u0026reg; environmental buffer, 250 nM of each sense and antisense primer (with the exception of the specific primer of \u003cem\u003eP. falciparum\u003c/em\u003e, labelled HEX, used at a concentration of 125 nM, see supplementary table 2. For each sample, two PET-PCR replicates were performed from 2 \u0026micro;L of template DNA. The thermocycling protocol included an initial activation at 95\u0026deg;C for 15 minutes, followed by 45 cycles including denaturation at 95\u0026deg;C for 20 seconds, then hybridization at 60\u0026deg;C for 40 seconds. A cycle threshold (Ct) of 35 was retained as the limit of positivity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetection of deletions of\u003c/b\u003e \u003cb\u003ethe Pfhrp2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePfhrp3 genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGenotyping of \u003cem\u003ethe Pfhrp2\u003c/em\u003e and \u003cem\u003ePfhrp3\u003c/em\u003e genes was performed by PCR uniplex (one-step), targeting exon 2 of each of the two genes. Published primers known as \"BRAVO\" (Supplementary table 3) were used, with optimized amplification conditions [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The presence of a deletion was concluded when no amplification product of the targeted gene was observed despite the successful completion of internal controls (amplification of the second \u003cem\u003ePfhrp gene\u003c/em\u003e or PET-PCR confirmation), thus excluding a technical failure.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe antigen detection data was processed and visualized in R (version 3.6.1) using ggplot2. The positivity threshold was determined for each antigen by the log-normal mean plus three standard deviations from a panel of 86 negative samples. Samples exhibiting an absent or significantly lower HRP2 signal and one or more other elevated antigens were grouped together as suspected of pfhrp2 gene deletion and selected for molecular analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAntigen screening\u003c/h2\u003e \u003cp\u003eA total of 438 DBS were analyzed by Luminex MAGPIX multiplex. The antigenic profiles identified samples suspected for \u003cem\u003ePfhrp2\u003c/em\u003e deletion. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the distribution of the different antigens compared with detection of the \u003cem\u003ePfhrp2\u003c/em\u003e antigen.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eConfirmation of\u003c/b\u003e \u003cb\u003eP. falciparum infections by PET-PCR\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmong the 438 patients included, 375 mono-infection of \u003cem\u003eP\u003c/em\u003e. \u003cem\u003efalciparum\u003c/em\u003e infections were confirmed by PET-PCR (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The remaining 63 samples were excluded, due to detected co-infections (\u003cem\u003eP. malariae\u003c/em\u003e, \u003cem\u003eP. ovale\u003c/em\u003e) or discrepancies between antigenic and parasitological results.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eDetection of\u003c/b\u003e \u003cb\u003ePlasmodial infections\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ePlasmodial\u003c/em\u003e species\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. falciparum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. falciparum\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eP. malariae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. falciparum\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eP. ovale\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. falciparum\u003c/em\u003e\u0026thinsp;+\u0026thinsp;\u003cem\u003eP. ovale\u0026thinsp;+\u0026thinsp;P. malariae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e438\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDeletion of\u003c/b\u003e \u003cb\u003ethe Pfhrp2\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ePfhrp3 genes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmong the 375 mono infection of \u003cem\u003eP. falciparum\u003c/em\u003e isolate, 2.9% (11/375) had a deletion of the \u003cem\u003epfhrp2 gene\u003c/em\u003e, 10.9% (41/375) had a deletion of the \u003cem\u003epfhrp3\u003c/em\u003e gene, and 1.3% (5/375) had a combined deletion of both genes. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e below summarizes the spatial distribution of these deletions. The frequencies of \u003cem\u003epfhrp2\u003c/em\u003e deletions ranged from 1.7% to 5.0% by site. Baban Tabki (Zinder) hab the high prevalence of \u003cem\u003epfhrp2\u003c/em\u003e. The \u003cem\u003ePfhrp3\u003c/em\u003e and double deletions \u003cem\u003ePfhrp2\u003c/em\u003e/\u003cem\u003ePfhrp3\u003c/em\u003e were found in all the study sites (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRegional distribution of \u003cem\u003epfhrp2\u003c/em\u003e and \u003cem\u003epfhrp3\u003c/em\u003e deletions among \u003cem\u003eP. falciparum\u003c/em\u003e infections (N\u0026thinsp;=\u0026thinsp;375)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStudy site (Region)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSamples\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003epfhrp2\u003c/em\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003epfhrp3\u003c/em\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003epfhrp2/pfhrp3\u003c/em\u003e (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAderbissinat (Agadez)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12 (15.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e2 (2.5%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAgui\u0026eacute; (Maradi)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e3 (3.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10 (10.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1 (1.0%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBaban Tabki (Zinder)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4 (5.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8 (10.0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1 (1.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBoboye (Dosso)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e115\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e2 (1.7%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11 (9.6%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1 (0.9%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11 (2.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41 (10.9%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5 (1.3%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eDeletions of the \u003cem\u003epfhrp2\u003c/em\u003e and \u003cem\u003epfhrp3\u003c/em\u003e genes represent a growing threat to the diagnostic performance of HRP2-based rapid tests, widely used for the detection of \u003cem\u003eP. falciparum\u003c/em\u003e in Africa. The present study identified the detection of these deletions in Niger. The analysis was carried out on samples derived from a therapeutic efficacy study (TES) conducted in Niger in 2022 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our study is the first to report the deletions of \u003cem\u003epfhrp2\u003c/em\u003e and \u003cem\u003epfhrp3\u003c/em\u003e in Niger, with a prevalence of 3% for \u003cem\u003epfhrp2\u003c/em\u003e and 10.9% for \u003cem\u003epfhrp3\u003c/em\u003e. The prevalence of \u003cem\u003epfhrp2 deletion\u003c/em\u003e remains below the 5%. The WHO recommends that countries consider switching away from exclusive reliance on HRP2-based tests when the rate of false-negative HRP2-based RDTs is greater or equal to 5% due to gene deletions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It\u0026rsquo;s important to higligh that our study was not initialy disease as described by WHO to determine the prevalence of pfhrp2/pfhp3 deletion. Based on the identification of \u003cem\u003epfhrp2\u003c/em\u003e and \u003cem\u003epfhrp3\u003c/em\u003e deletions in Niger, further studies are required to determine the false negative rate of HRP2-based based on the WHO protocole.\u003c/p\u003e \u003cp\u003eFurthermore, a geographical variation in deletion frequency was observed. The specific prevalence of the pfhrp2 deletion in Zinder is 5.0%. The heterogeneous distribution observed between the sites argues for distinct local dynamics. At Zinder, the high rate of \u003cem\u003epfhrp2-deletion\u003c/em\u003e could be related to proximity to northern Nigeria, where similar rates have been reported [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], suggesting a transmission of deleted parasites across national boundaries or shared selection pressure. In contrast, the situation in Agadez, a desertic region with low transmission, could reflect a founder effect or genetic drift in an isolated population [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In-depth genomic analysis (microsatellites, WGS sequencing) would provide a better understanding of the emergence and dispersion of these variants [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast to our study, the highest prevalences were reported in Ethiopia (5.75% for \u003cem\u003epfhrp2\u003c/em\u003e, 35.53% for \u003cem\u003epfhrp3\u003c/em\u003e) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and in some regions of East Africa [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], while several countries, including Togo [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and Mozambique, reported no deletions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Most West and Central Africa still reports sporadic or absent deletions [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/p\u003e \u003cp\u003eRegarding deletions of the \u003cem\u003ePfhrp3\u003c/em\u003e gene, their high prevalence (up to 15% in Agadez) is part of a global trend observed in other African contexts, where losses of \u003cem\u003ePfhrp3\u003c/em\u003e seem to occur more frequently than those of \u003cem\u003ePfhrp2\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Parasites carrying combined deletions of both genes (1.3% in our study) are of particular concern, as they completely evade HRP2 RDTs, exposing patients to a risk of serious false negatives [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFrom a public health perspective, the continued use of HRP2-based RDTs remains justified at the national level. However, the presence of critical foci such as the one identified in Zinder calls for additional targeted investigations, particularly on symptomatic RDT-negative cases. If a prevalence of false-negative HRP2-based RDTs\u0026thinsp;\u0026ge;\u0026thinsp;5% is confirmed, the adoption of alternative diagnostic tests, such as dual-antigen HRP2/pLDH or RDTs targeting pan-plasmodial LDH or PfLDH antigens should be considered in circumstances where microscopy is not feasible, in line with WHO recommendations [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne limitation of this study was the use of samples which tested positive by both microscopy and HRP2-based RDT, potentially biasing the selection of samples to include wildtype \u003cem\u003epfhrp2/3\u003c/em\u003e parasites. However, the fact that some \u003cem\u003ePfhrp2-negative\u003c/em\u003e parasites were still detected despite initial screening based on the HRP2 RDT suggests that the closely-related HRP3 protein may partially compensate for the absence of HRP2 due to the presence of similar repeat epitopes present in both proteins [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This phenomenon complicates the identification of deleted strains [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Conversely, it is also likely that some infections with parasites carrying \u003cem\u003ePfhrp2-deletion\u003c/em\u003e were wrongly excluded at the screening stage, due to a lack of RDT positivity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. This means that the true prevalence may be underestimated in our study. Another limitation is the antigen screening using multiplex bead-based assays used to prioritize samples for molecular genotyping; while this approach improves efficiency, it may fail to identify low-density infections or parasites with residual HRP2 or compensatory HRP3 expression, potentially leading to misclassification. Finally, although sentinel sites were selected to represent distinct epidemiological areas, the findings may not be fully generalizable to all regions of the country, particularly areas with different transmission intensities or diagnostic practices.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides the first molecular data on the presence of deletions of the \u003cem\u003epfhrp2\u003c/em\u003e and \u003cem\u003epfhrp3\u003c/em\u003e genes in \u003cem\u003eP. falciparum\u003c/em\u003e in Niger. The prevalence of \u003cem\u003epfhrp2\u003c/em\u003e deletion remains below 5% among all samples. The higher frequency of \u003cem\u003epfhrp3\u003c/em\u003e deletion and the identification of double-deletion strains reinforce the need for surveillance. Our findings support the establishment of an integrated molecular surveillance system, including genotyping of RDT-negative/microscopy positive cases, to anticipate diagnostic failures and adapt national malaria control policies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eACTs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eArtemisinin-based Combination Therapies\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eAW1 / AW2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWash buffers\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003ebp\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBase pairs\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eCDC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eU.S. Centers for Disease Control and Prevention\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eCERMES\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCentre de Recherche M\u0026eacute;dical et Sanitaire (Niamey, Niger)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eCIGASS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInternational Research and Training Center in Applied Genomics and Health Surveillance (Dakar, S\u0026eacute;n\u0026eacute;gal)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eCNERS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eComit\u0026eacute; National d\u0026rsquo;\u0026Eacute;thique pour la Recherche en Sant\u0026eacute; (Niger)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eCt\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCycle threshold\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eDBS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDried blood spots\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eDNA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDeoxyribonucleic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eELISA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eEnzyme-Linked Immunosorbent Assay\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eHRP2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHistidine-rich protein 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eHRP3\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHistidine-rich protein 3\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eMSP1 / Pfmsp1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMerozoite surface protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eMSP2 / Pfmsp2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMerozoite surface protein 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003epAldolase\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePan-plasmodial aldolase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003ePCR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePolymerase Chain Reaction (r\u0026eacute;action de polym\u0026eacute;risation en cha\u0026icirc;ne)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003ePET-PCR\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhoto-induced electron transfer - Polymerase Chain Reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003epfhrp2 / pfhrp3\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eG\u0026egrave;nes codant pour les prot\u0026eacute;ines HRP2 et HRP3 chez \u003cem\u003eP. falciparum\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003epfLDH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eP. falciparum\u003c/em\u003e lactate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003epLDH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePan-specific lactate dehydrogenase (lactate d\u0026eacute;shydrog\u0026eacute;nase commune aux esp\u0026egrave;ces de \u003cem\u003ePlasmodium\u003c/em\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003ePMI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eU.S. President's Malaria Initiative\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003ePNLP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProgramme National de Lutte contre Paludisme (Niger)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003epoLDH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eP. ovale\u003c/em\u003e lactate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003ePvLDH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eP. vivax\u003c/em\u003e lactate dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eRDTs\u003c/b\u003e (TDR)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRapid diagnostic tests\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eTES\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTherapeutic efficacy study\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eUAS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eUniversit\u0026eacute; Andr\u0026eacute; Salifou (Zinder, Niger)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eUSAID\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eU.S. Agency for International Development\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eWGS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWhole Genome Sequencing\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u0026bull; \u003cb\u003eWHO\u003c/b\u003e (OMS)\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWorld Health Organization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eThis study received authorization from the National Ethics Committee for Health Research (CNERS) on 12/07/2022 by letter referenced N\u0026deg;32/2022/CNERS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e All data generated or analysed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that there is no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was funded by\u0026nbsp;the U.S. President\u0026apos;s Malaria Initiative Impact Malaria, Niger.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contribution:\u0026nbsp;\u003c/strong\u003eI.M.L., I.I. and MKS: Contributed to the writing of the protocol, its implementation, the writing of the manuscript and the management of the teams. I.I and MML write the first and final draft of the manuscript. II and MKS: Participated in the collection, data analysis, molecular study in Senegal, and in the drafting of the report. A.B.D; M.A.D; D.S; J.M; I.C and AS: led the training and technical supervision of the researchers during the molecular analysis and also participated in the data analysis and writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u0026nbsp;\u003c/strong\u003eWe would like to thank Prof. Daouda Ndiaye for having received the Nigerien teams in his CIGASS laboratory for the molecular analyses of \u003cem\u003epfhrp2\u003c/em\u003e/3. We thank Dr Irene Cavros and Cassandra Webster for the support at the PARMA Hub\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organization. 2023; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://creativecommons.org/licenses/by-nc-sa/3.0/igo\u003c/span\u003e\u003cspan address=\"https://creativecommons.org/licenses/by-nc-sa/3.0/igo\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalaria facts \u0026amp; statistics. 2025 [Internet]. Med. Malar. Venture. 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Malaria control and the unexpected spread of diagnostic-resistant Plasmodium falciparum in Peru [Internet]. bioRxiv; 2026 [cited 2026 Mar 11]. p. 2026.02.25.707493. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.64898/2026.02.25.707493\u003c/span\u003e\u003cspan address=\"10.64898/2026.02.25.707493\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"P. falciparum, Pfhrp2/3, HRP2-based RDTs, gene deletion, Malaria diagnostic, Niger","lastPublishedDoi":"10.21203/rs.3.rs-9407357/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9407357/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRapid diagnostic tests (RDTs) based on histidine-rich protein 2 (HRP2) are the main diagnostic tool for malaria in Niger and many countries in sub-Saharan Africa. However, deletions of the \u003cem\u003eP. falciparum hrp2\u003c/em\u003e and \u003cem\u003ehrp3\u003c/em\u003e genes can compromise RDT performance, and pose a threat to diagnostic accuracy. Although these deletions were reported in several regions of Africa, Asia and South America, no molecular data on \u003cem\u003epfhrp2/3\u003c/em\u003e were previously available for Niger.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe present study is a retrospective sub-analysis of biological samples derived from a therapeutic efficacy study (TES) conducted in Niger in 2022. Antigen profiling was performed using a multiplex bead-based assay to identify samples with weak or undetectable HRP2 signal. Species confirmation was conducted by PET-PCR, and \u003cem\u003epfhrp2\u003c/em\u003e/3 exon 2 genotyping was performed using one-step PCR.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eAmong the 375 \u003cem\u003eP. falciparum\u003c/em\u003e mono-infection isolates analyzed, \u003cem\u003ePfhrp2\u003c/em\u003e deletions were found in 11/375 (3.0%), \u003cem\u003ePfhrp3\u003c/em\u003e deletions in 41/375 (10.9%), and double deletions in 5/375 (1.3%) samples. The highest prevalence of site-specific \u003cem\u003ePfhrp2\u003c/em\u003e deletions (5.0%; 4/80) was observed at Baban Tabki (Zinder).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis is the first molecular evidence of \u003cem\u003ePfhrp2\u003c/em\u003e and \u003cem\u003ePfhrp3\u003c/em\u003e deletions in \u003cem\u003eP\u003c/em\u003e in Niger. The \u003cem\u003ePfhrp2\u003c/em\u003e deletion rate remains below the 5% threshold set by WHO for the revision of the RDT policy. It\u0026rsquo;s important to use WHO protocole for \u003cem\u003ePfhrp2\u003c/em\u003e/3 deletion to determine the national prevalence of these genes deletion in Niger.\u003c/p\u003e","manuscriptTitle":"First detection of pfhrp2 and pfhrp3 gene deletions in Niger Republic: a retrospective sub- analysis of biological samples","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-26 17:10:43","doi":"10.21203/rs.3.rs-9407357/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-22T14:58:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"127961997094116225450764584535442522670","date":"2026-04-22T10:58:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38471049498479370484894677457538973799","date":"2026-04-19T12:30:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-17T10:42:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-15T10:59:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-15T10:58:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Malaria Journal","date":"2026-04-13T17:51:13+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b6f5aba-57a4-4287-878a-821411f8877d","owner":[],"postedDate":"April 26th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-26T17:10:43+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-26 17:10:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9407357","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9407357","identity":"rs-9407357","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}