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

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 3% (11/375; 95% CI 1–5%), pfhrp3 deletions in 11% (41/375; 95% CI 8–14%), and double deletions in 1% (5/375; 95% CI 0–2%). These figures likely represent a conservative estimate, as the true prevalence may be higher due to the antigen pre-screening and the selection bias inherent in the original study design.

Conclusions

This is the first molecular evidence of Pfhrp2 and Pfhrp3 deletions in P. falciparum in Niger. The observed pfhrp2 deletion rate is a significant molecular finding; however, further evidence is needed to determine if the rate of false-negative RDTs caused by these deletions reaches the 5% threshold set by WHO for a national RDT policy revision.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12936-026-06069-7.

Introduction

Malaria remains a critical public health challenge in sub-Saharan Africa, where Plasmodium falciparum (P. falciparum) is responsible for the majority of cases and deaths [1]. In 2024, Niger accounted for approximately 6.1% of all malaria-related deaths in the WHO African Region, ranking third among the most affected countries [2]. Effective malaria control in Niger relies on rapid, accurate diagnosis followed by prompt treatment to reduce morbidity and mortality. While P. falciparum is the dominant species, other species including P. vivax, P. ovale, and P. malariae also circulate, complicating diagnosis and management due to their potential for relapses and differing drug resistance profiles [3]. Global control efforts have significantly reduced the malaria burden through the scale-up of insecticide-treated nets, indoor residual spraying, and artemisinin-based combination therapies (ACTs) [4, 5 ]. Central to these efforts is the widespread adoption of rapid diagnostic tests (RDTs). In Niger, over 90% of malaria diagnoses are performed using RDTs, specifically those targeting the histidine-rich protein 2 (HRP2) [6, 7 ]. These tests are preferred in resource-limited and rural settings due to their ease of use, low cost, and superior performance compared to pLDH-based RDTs.

However, the reliability of HRP2-based RDTs is increasingly threatened by the emergence of P. falciparum parasites with deletions in the pfhrp2 and pfhrp3 genes, which encode the HRP2 and HRP3 proteins. These deletions can result in false-negative RDT results, even in patients with high parasitemia. First identified in 2010 in South America [8], these deletions have since been reported globally, with prevalence rates exceeding 5% in several African countries, including Eritrea, Djibouti, and South Sudan [9–12]. To manage this threat, the WHO recommends transitioning to alternative diagnostic tools, such as pLDH-based or dual-antigen RDTs, when the rate of false-negative HRP2-RDTs due to these deletions reaches ≥ 5% [13].

In Niger, the status of pfhrp2/3 gene deletions remained unknown due to a lack of molecular surveillance data. Given the national health system's heavy reliance on HRP2-based RDTs, assessing the presence of these deletions is urgent. This study aims to provide the first molecular evidence of pfhrp2 and pfhrp3 deletions in Niger by performing a retrospective sub-analysis of samples collected during a 2022 multicenter therapeutic efficacy study.

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 genomic analysis of pfhrp2 and pfhrp3 gene deletions, inclusion was restricted to Day 0 (pre-treatment) samples. This restriction was implemented to ensure that the molecular characterization reflects the baseline parasite population prior to drug exposure. Furthermore, Day 0 corresponds to the point of maximum parasitemia for each patient, thereby ensuring optimal DNA yield and higher sensitivity for the molecular detection of gene deletions. Four sentinel sites Aderbissinat (Agadez), Aguié (Maradi), Baban Tabki (Zinder), and Boboye (Dosso) were selected to ensure geographical and epidemiological representativeness across Niger’s diverse malaria landscapes, covering northern desertic regions with low transmission and southern areas with higher seasonal transmission.

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]. Several controls were utilized to exclude false-negative results. DNA integrity was verified by the successful amplification Pfmsp1 and Pfmsp2 and the species-specific PET-PCR. Only samples with confirmed amplifiable parasite DNA were included in the deletion analysis. Furthermore, each PCR batch included a positive control and a no-template negative control to monitor for amplification efficiency and potential reagent contamination.

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 min, followed by 45 cycles including denaturation at 95 °C for 20 s, then hybridization at 60 °C for 40 s. 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]. Genotyping of the pfhrp2 and pfhrp3 genes was performed by one-step PCR targeting specifically the exon 2 of each gene. While exon 2 encodes the majority of the protein and includes the repeat epitopes targeted by HRP2-based RDTs, this approach is a limitation as it cannot detect partial deletions or mutations occurring outside of this region. To ensure the accuracy of deletion calls, all isolates initially showing an absence of amplification for pfhrp2 or pfhrp3 were re-tested in duplicate or triplicate. A gene deletion was definitively concluded only when at least two independent PCR reactions failed to produce the expected amplicon, while internal controls were successfully amplified. In the event of discordant results between replicates, the sample was re-analyzed or excluded to prevent false-positive reporting. 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 for each antigen (HRP2, pfLDH, pLDH, and pAldolase) was operationally defined as the log-normal mean plus three standard deviations (SD), established from a reference panel of 86 malaria-negative samples. A sample was quantitatively classified as having a 'low or absent HRP2 signal’ if its Median Fluorescent Intensity (MFI) fell below this calculated threshold. To prioritize samples for molecular analysis, a discordant antigenic profile was required, defined as an HRP2 signal below the threshold in the presence of one or more other antigens (e.g., pAldolase or pfLDH) with signals exceeding their respective thresholds. To compare the prevalence of pfhrp2 and pfhrp3 deletions across the four sentinel sites, Pearson’s Chi-square test was employed with 3 degrees of freedom (df). All statistical tests were two-sided, and a p-value < 0.05 was considered statistically significant.

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.

Fig. 1.

Distribution of the different antigens according to the Pfhrp2 antigen. Scatter plots comparing HRP2 Median Fluorescent Intensity (MFI) against three reference antigens: A pfLDH, B pAldolase, and C pLDH for 438 DBS samples. The horizontal and vertical dashed lines represent the statistically defined positivity thresholds (mean + 3 SD). The highlighted ‘Discordant Zone’ (lower right quadrant) identifies samples with low or absent HRP2 signal despite high levels of other Plasmodium antigens, which were prioritized for molecular genotyping to confirm pfhrp2 deletions

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 (Supplementary Fig. 1).

Table 1.

Detection of Plasmodial infections

Plasmodial species	n	%	IC95%
P. falciparum	375	86	82–89
P. falciparum + P. malariae	30	7	4–9
P. falciparum + P. ovale	20	5	3–7
P. falciparum + P. ovale + P. malariae	13	3	1–5
Total	438	100

Deletion of the Pfhrp2 and Pfhrp3 genes

Among the 375 mono infection of P. falciparum isolate, 3% (11/375; IC95%:1–5)) had a deletion of the pfhrp2 gene, 11% (41/375; IC95%:8–14) had a deletion of the pfhrp3 gene, and 1% (5/375; IC95%:0–2) 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) exhibited the highest site-specific prevalence of pfhrp2. The Pfhrp3 and double deletions Pfhrp2/Pfhrp3 were found in all the study sites (Table 2). No statistically significant difference in deletion prevalence was observed between the four study sites for either pfhrp2 (χ2 = 1.83, p = 0.608) or pfhrp3 (χ2 = 1.74, p = 0.628) (Fig. 2).

Table 2.

Regional distribution of pfhrp2 and pfhrp3 deletions among P. falciparum infections (N = 375)

Study site (Region)	Samples	pfhrp2 (%)	IC95%	pfhrp3 (%)	IC95%	pfhrp2/pfhrp3 (%)	IC95%
Aderbissinat (Agadez)	80	2 (3%)	0–6	12 (15%)	7–23	2 (3%)	0–6
Aguié (Maradi)	100	3 (3%)	0–6	10 (10%)	4–16	1 (1%)	0–3
Baban Tabki (Zinder)	80	4 (5%)	0–10	8 (10%)	3–17	1 (1%)	0–4
Boboye (Dosso)	115	2 (2%)	0–4	11 (10%)	4–15	1 (1%)	0–3
Total	375	11 (2.9%)	1–5	41 (11%)	8–14	5 (1%)	0–3

Fig. 2.

Distribution of pfhrp2 and pfhrp3 gene deletion profiles across study sites. Stacked bars represent the proportion of Plasmodium falciparum isolates with a pfhrp2 deletion only (blue), a pfhrp3 deletion only (orange), a combined pfhrp2/pfhrp3 deletion (green), or no deletion (gray) at the four study sites: Aderbissinat (Agadez, n = 80), Aguie (Maradi, n = 100), Baban Tabki (Zinder, n = 80), and Boboye (Dosso, n = 115). The percentages displayed within the gray segments indicate the proportion of isolates without gene deletions at each site. Overall, the majority of isolates lacked pfhrp2 and/or pfhrp3 deletions, with frequencies ranging from 80.0% to 87.8% across the study locations (Figure 2)

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. It is crucial to distinguish this from the clinical false-negative rate. The WHO recommends a change in diagnostic policy only when the prevalence of HRP2-based RDT false-negative results caused by gene deletions is ≥ 5% [23]. It is important to highlight that our study was not initially designed as described by WHO to determine the prevalence of pfhrp2/pfhrp3 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 protocol.

Furthermore, a geographical variation in deletion frequency was observed. The specific prevalence of the pfhrp2 deletion in Zinder is 5.0%, while this regional variation was not statistically significant when compared to the other sites. The heterogeneous distribution observed between the sites argues for distinct local dynamics. In Zinder, the observed pfhrp2 deletion rate represents a hypothesis for further investigation given the region’s proximity to northern Nigeria, where comparable deletion rates have been documented [24]. While this geographic proximity may suggest potential transborder transmission or shared selection pressures, our current data do not allow for a definitive conclusion. In-depth genomic analysis (microsatellites, WGS sequencing) would provide a better understanding of the emergence and dispersion of these variants.

Our findings in Niger align with the broader trends observed in West and Central Africa, where pfhrp2 and pfhrp3 deletions are generally reported as sporadic or absent [12, 24, 31 –]. In contrast, significantly higher prevalence rates have been documented across East Africa and the Horn of Africa, particularly in Ethiopia [26] (5.75% for pfhrp2 and 35.53% for pfhrp3) and other countries in the region that have already exceeded the 5% WHO threshold for diagnostic policy revision [27].

The high prevalence of pfhrp3 deletions (overall 11% and 15% in Agadez) carries significant indirect implications for malaria diagnosis in Niger. Due to the high structural homology between the HRP2 and HRP3 proteins, most HRP2-based RDTs exhibit cross-reactivity with HRP3 epitopes. In many cases, HRP3 expression can partially compensate for the loss of pfhrp2, allowing a parasite to still be detected by a standard RDT, albeit with reduced sensitivity [32]. Therefore, the 11% pfhrp3 deletion rate we report should be viewed as a precursor to diagnostic vulnerability, predisposing the parasite population to rapid diagnostic escape if pfhrp2 deletions continue to spread. Parasites carrying combined deletions of both genes (1% 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 studied sentinel sites. 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].

A primary limitation of this study is the inherent selection bias associated with the original TES design, which only enrolled symptomatic patients who were HRP2-RDT positive. Consequently, parasites with double pfhrp2/3 deletions, which typically yield negative RDT results, were likely excluded at the recruitment stage unless compensatory HRP3 expression allowed for RDT positivity [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. Furthermore, the antigen-first screening strategy used to prioritize samples for molecular analysis may have introduced a classification bias. By only genotyping isolates with ‘suspected’ antigenic profiles (low or absent HRP2 signal), we may have missed pfhrp2-deleted parasites that still produced enough protein (either residual HRP2 or through HRP3 compensatory expression) to exceed our multiplex positivity threshold. Additionally, low-density infections where antigens are below the Luminex detection limit might have been misclassified. As a result, the molecular prevalence of 3.0% for pfhrp2 reported here likely represents an underestimation of the true prevalence in the parasite population. Future surveillance should ideally include direct molecular screening of a random subset of all samples, regardless of their HRP2 status, to bypass this bias. 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. While the molecular prevalence of pfhrp2 deletions remains below 5% at the studied sentinel sites, these results provide the first evidence for the need to monitor the diagnostic performance of RDTs, particularly in high-prevalence areas like Zinder, to ensure the WHO threshold for false-negatives is not exceeded. 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

Author contributions

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.

Funding

This study was funded by the U.S. President’s Malaria Initiative Impact Malaria, Niger.

Data availability

No datasets were generated or analysed during the current study.

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.

Competing interests

The authors declare no competing interests.