Plasmodium falciparum is evolving to escape malaria rapid diagnostic tests in Ethiopia

In Africa, most rapid diagnostic tests (RDTs) for falciparum malaria recognize histidine-rich protein 2 antigen. Plasmodium falciparum parasites lacking histidine-rich protein 2 (pfhrp2) and 3 (pfhrp3) genes escape detection by these RDTs, but it is not known whether these deletions confer sufficient selective advantage to drive rapid population expansion. By studying blood samples from a cohort of 12,572 participants enroled in a prospective, cross-sectional survey along Ethiopia’s borders with Eritrea, Sudan and South Sudan using RDTs, PCR, an ultrasensitive bead-based immunoassay for antigen detection and next-generation sequencing, we estimate that histidine-rich protein 2-based RDTs would miss 9.7% (95% confidence interval 8.5–11.1) of P. falciparum malaria cases owing to pfhrp2 deletion. We applied a molecular inversion probe-targeted deep sequencing approach to identify distinct subtelomeric deletion patterns and well-established pfhrp3 deletions and to uncover recent expansion of a singular pfhrp2 deletion in all regions sampled. We propose a model in which pfhrp3 deletions have arisen independently multiple times, followed by strong positive selection for pfhrp2 deletion owing to RDT-based test-and-treatment. Existing diagnostic strategies need to be urgently reconsidered in Ethiopia, and improved surveillance for pfhrp2 deletion is needed throughout the Horn of Africa.

P lasmodium falciparum strains that evade diagnosis by RDTs represent a major threat to malaria control and elimination efforts 1,2 . Malaria RDTs detect antigens produced by Plasmodium parasites, including P. falciparum histidine-rich protein 2 (HRP2), parasite lactate dehydrogenase (LDH) and aldolase. HRP2 has advantages over other biomarkers because of its abundance in the bloodstream, repetitive binding epitopes and falciparum-specificity [3][4][5] . Most HRP2-based RDTs also exhibit some cross-reactivity to a closely related protein (HRP3) 6 . Of the 345 million RDTs sold annually, HRP2-based RDTs are the predominant malaria diagnostic test, the majority of which are deployed throughout sub-Saharan Africa 7 .
Deletion mutations involving the histidine-rich protein 2 and/ or 3 (pfhrp2/3) genes allow parasite strains to escape HRP2-based RDT detection 8,9 . First described in clinical samples from Peru in 2010, these subtelomeric deletions on chromosomes 8 (pfhrp2) and 13 (pfhrp3) are frequently large (≥20 kb), encompass multiple genes and are difficult to study using existing methods 8,10 . Improved PCR and serological approaches can be used to increase confidence in deletion prevalence estimates [11][12][13] , but our understanding of the evolutionary history of pfhrp2/3-deleted P. falciparum is limited and largely informed by analysis of a small number of microsatellite markers [14][15][16] . Recent genomic analyses have begun to expand our understanding of pfhrp2/3-deleted P. falciparum [17][18][19] but continue to be hindered by the challenges of assembling the highly repetitive and paralogous sequences of P. falciparum subtelomeres 20 .
New tools are needed to support surveillance of pfhrp2/3 deletions, determine their true prevalence and understand the forces impacting their evolution and spread.
'Diagnostic-resistant' pfhrp2/3-deleted parasites have now been observed in multiple sites across Africa. Reports from 2017-2018 prompted calls for urgent surveillance in affected regions, including countries in the Horn of Africa like Ethiopia 14,[21][22][23][24][25][26] . Ethiopia is Africa's second most-populous country, and around 60% of its population is at risk of malaria exposure 27 . Plasmodium falciparum infection accounts for the majority of malaria deaths and approximately 70% of all cases 27,28 . RDTs were first introduced in Ethiopia in 2004, and the country's current test-treat-track strategy requires parasitological confirmation either by quality microscopy or RDT before antimalarial treatment 29 . Plasmodium falciparum-Plasmodium vivax (HRP2/Pv-specific-LDH) combination RDTs are the sole diagnostic test used in most settings. Over the past decade, Ethiopia has achieved remarkable progress in the fight against malaria through strong preventative and case management interventions, including engagement of health extension workers to provide diagnostic services at a local level 29 . Reports of highly prevalent pfhrp2/3-deleted parasites in neighbouring Eritrea suggest that these gains could be threatened 14,22 . Rapid assessment of the epidemiology of pfhrp2/3 deletions in Ethiopia and surrounding regions is required to determine whether a change in malaria diagnostic testing policy is warranted.
Using the largest prospective study of pfhrp2/3-deleted P. falciparum performed so far, we apply genomic tools to determine the genetic epidemiology of pfhrp2/3-deleted P. falciparum in sites spanning Ethiopia's borders with Eritrea, Sudan and South Sudan. In this study, based on the World Health Organization's (WHO) pfhrp2/3 deletion surveillance protocol released in 2018 to encourage a harmonized and representative approach to pfhrp2/3 deletion surveillance and accurate reporting 30 , we confirm deletions using multiple PCR assays 13 , an ultrasensitive bead-based immunoassay for antigen detection 12 , whole-genome sequencing (WGS) 31,32 and/ or molecular inversion probe (MIP) deep sequencing 33 . Using a new MIP panel designed for high-throughput pfhrp2/3 genotyping, we map and categorize deletion breakpoints and evaluate their flanking regions for evidence of recent evolutionary pressure favouring pfhrp2/3-deleted parasites.
Pfhrp2/3 deletion PCR genotyping. Eight hundred and twenty samples with complete demographic and clinical data from Amhara (n = 524), Tigray (n = 225) and Gambella (n = 71) underwent molecular analysis. These samples were collected from participants with the discordant RDT profile and a subset of participants with other RDT results ( Fig. 2 and Supplementary Note). Further analysis was restricted to the 610 samples with >100 parasites per µl to avoid misclassification of pfhrp2/3 deletions due to low parasitaemia (Extended Data Fig. 1); 176 samples (28.9%) had the discordant RDT profile.
Genetic signatures of evolutionary selection. Extended haplotype homozygosity (EHH) statistics revealed signatures of recent positive selection in the flanking region centromeric to pfhrp2 deletions on chromosome 8 but not in flanking regions around pfhrp3 deletions on chromosome 13 (ref. 36   with the pfhrp2 deletion (0.968) along the entire 28 kb analysed, whereas homozygosity around the pfhrp2-intact (wild-type) allele quickly broke down (Fig. 5a). A similar pattern was observed when deletion profiles were analysed separately; chr8-P4 EHH remained high and chr8-P1-P3 EHH broke down quickly (Extended Data Fig.  8). We further confirmed high EHH around the pfhrp2 deletion allele using WGS data. Comparing 23 whole-genome sequenced samples from this study and the 25 published MalariaGEN samples described above, we were able to extend our analysis and confirm an EHH length of >143 kb centromeric to the deletion (Extended Data  Fig. 9a). These findings suggest a recent selective sweep, indicative of strong evolutionary pressure favouring pfhrp2-deleted P. falciparum parasites.
A different pattern was observed in the regions flanking pfhrp3 (Fig. 5b). EHH quickly decreased below 0.5 for pfhrp3 deletion alleles as well as the pfhrp3-intact allele within 1 kb of available single-nucleotide polymorphisms. When deletion profiles were analysed as separate alleles, the EHH pattern was similarly low for chr13-P1, -P3 and -P4 (Extended Data Fig. 10). Comparison of EHH around the pfhrp3-intact and P3-like pfhrp3 deletions using WGS data from the 25 MalariaGEN samples confirmed our finding that the EHH decreased quickly for both pfhrp3-intact and pfhrp3 deletion alleles (Extended Data Fig. 9b). Taken together, these findings suggest that each pfhrp3 deletion profile arose multiple times independently, and/or they have been present in the parasite population for sufficient time for homozygosity due to genetic hitchhiking to be degraded by recombination with different haplotypes.

Discussion
Using the largest prospective study of pfhrp2/3-deleted P. falciparum performed so far and complementary molecular, immunological and sequencing assays, we provide clear evidence that pfhrp2/3-deleted parasites are circulating in multiple sites along Ethiopia's borders with Sudan and Eritrea. Analysis of flanking haplotypes suggests that the pfhrp2 deletion mutation emerged and recently expanded from a single origin, whereas pfhrp3 deletion mutations have existed for a longer time span and likely have multiple origins. As expected, we did not observe perfect concordance between RDT results, PCR, a bead-based immunoassay, WGS and MIP sequencing results. However, the preponderance of evidence from these diverse platforms provides robust confirmation of deletions and supports use of the WHO protocol for rapid pfhrp2/3 deletion surveillance 30 . This protocol provides standardized data collection, fieldwork and sampling methods that can be adapted to the local context and is intended to help programmes rapidly determine whether deployment of alternative diagnostics is needed. The prevalence of false-negative HRP2-based RDT results due to pfhrp2 deletions is estimated at 9.7% overall and up to 11.5% and 14.9% in the Amhara and Tigray regions, respectively. These estimates exceed WHO minimum criteria (>5%) for a change in national diagnostic testing strategy. Pfhrp2/3-deleted parasites threaten recent progress made by Ethiopia's malaria control and elimination programme, and raise concerns about the ongoing use of and exclusive reliance on HRP2-based RDTs in the region for falciparum malaria diagnosis. However, transition to alternative combination diagnostics is not straightforward given the poor performance of Pf-LDH-based RDTs during multiple rounds of WHO product testing and versus PCR in this study (Supplementary Note  and Supplementary Table 11) and the challenges of conducting high-quality microscopy in the field 37 . Currently, only one combination Pf-LDH/Pv-LDH product suitable for Ethiopia is approved for purchase using Global Fund financing (https://www.theglobalfund.org/media/5891/psm_qadiagnosticsmalaria_list_en.pd f?u=636438486100000000).
Eritrea's alarming reports of false-negative RDTs due to pfhrp2/3-deleted parasites prompted an immediate change in national diagnostic testing policy in 2016 14,38 . Recent evidence from Sudan, Djibouti and Somalia suggests that the Horn of Africa may already be heavily affected by pfhrp2/3-deleted parasites 39,40 , although results from ongoing surveillance efforts are not yet publicly available. Within affected regions in Ethiopia, we observed spatial heterogeneity in P. falciparum RDT profiles by district, with prevalence of the discordant HRP2−, Pf-LDH+ RDT profile ranging from 1 to 30% (Supplementary Table 2). Although finer scale spatial analyses were not possible because of our health facility sampling approach, this finding is consistent with prior studies showing variation within countries and by region 23 . Differences in transmission intensity, treatment-seeking behaviour, diagnostic testing capacity and seasonality may account for some of the spatial variation in pfhrp2/3 deletion prevalence estimates 16,[41][42][43] . Although the factors driving emergence of these parasites in some regions but not others remain poorly understood, our study suggests that pfhrp2-deleted parasites may have spread widely within Ethiopia from a single origin. This finding is consistent with early microsatellite analysis of pfhrp2/3-deleted strains in Eritrea, in which 30 of 31 (96.8%) pfhrp2-deleted strains fell into a single genetically related cluster 14 , and raises concern about clonal expansion of pfhrp2-deleted strains in the Horn of Africa.
Using a multifaceted approach, we validate the use of MIP sequencing for high-throughput pfhrp2/3 deletion genotyping, deletion profiling and population genetic analysis. Comparison of MIP sequencing with other approaches demonstrated that it can be used for cost-effective (approximately US$10-15 per sample) and scalable deletion genotyping in samples with parasite densities of approximately 1,000 parasites per µl. Although this threshold can probably be improved by additional sequencing of samples with inadequate sequencing depth-of-coverage, in this case, the equivalent of half a NextSeq 550 flow cell enabled visualization of deletion breakpoint regions and variant calling in P. falciparum's subtelomeres in a large portion of samples, without the need for costly enrichment and WGS.
Based on analysis of MIP sequencing and available WGS data, we posit one potential model by which pfhrp2/3-deleted parasite populations may have evolved in the Horn of Africa. Findings from this study suggest that parasite populations with pfhrp3 deletions expanded in the more distant past and potentially arose multiple times independently, based on low EHH surrounding pfhrp3, multiple deletion profile patterns, the high overall frequency of pfhrp3-deleted parasites and their presence in older samples from 2013 in the MalariaGEN study. In this milieu, recent strong selection favouring parasites with deletions of pfhrp2 probably occurred due to 'test-track-treat' policies that rely on HRP2-based RDTs and allow parasites with deletions of both genes, or in some cases one of the two genes, to escape treatment. Implicit in this model is the assumption that forces apart from RDT-derived pressure are also driving the evolution of pfhrp2/3 deletions. Malaria due to pfhrp2+/3− parasites should be detectable by HRP2-based RDTs 44 . Further, pfhrp2/3-deleted parasites are highly prevalent in South America 23 , where RDT-based treatment decisions have never been common.
What other advantages might pfhrp2/3-deleted parasites have over those with intact genes? Our limited understanding of the biology of these deletions makes this question hard to answer. Several lines of enquiry may be relevant. (1) They may be better adapted to low transmission intensity settings than other strains. Pfhrp2/3-deleted parasites appear to be more common in regions with lower transmission and, presumably, lower complexities of infection, as in the current study 42 . However, this trend could also be an artefact of the assays used to detect them, that is, neither PCR, antigen immunoassays nor common sequencing methodologies are well suited to detect a pfhrp2/3-deleted strain when pfhrp2/3-intact strains have co-infected a human host. (2) Loss of pfhrp2/3 or flanking genes may alter parasite virulence. Evidence is accumulating that HRP2 plays a role in cerebral malaria and endothelial inflammation during severe malaria 45,46 . People infected by pfhrp2/3-deleted parasites may have less severe disease and therefore be less likely to seek treatment, increasing the likelihood of onward transmission. However, we cannot exclude the possibility that pfhrp2/3 are lost as  (55) 14 (13) 11 (37) WGS, n (%) a 0 22 0 a consequence of selection on other genes. For example, the flanking gene EBL-1 is almost uniformly lost in pfhrp3-deleted parasites in this cohort and appears to play a role during invasion of red blood cells 35,47 . Similarly, members of the rifin and stevor gene families with potential roles in parasite virulence were lost in the subtelomeric deletions observed in this study 48,49 . We did not observe evidence of an association between virulence and subtelomeric deletions in our cohort, but limited clinical data prevents us from assessing the hypothesis rigorously. (3) Loss of pfhrp2/3 or flanking genes may improve transmissibility to or from mosquitoes. To our knowledge, this phenomenon has not been studied. These and other hypotheses require experimental and improved epidemiological analyses. Regardless of the evolutionary forces at play, our findings strongly suggest that the evolution of pfhrp2/3-deleted parasites in Ethiopia was a multistep process that involved earlier expansion of pfhrp3than pfhrp2-deleted parasite populations. This study has several limitations. First, the study design prioritized evaluation of samples with discordant RDT results (HRP2− but Pf-LDH+) for rapid assessment of false-negative RDTs due to pfhrp2/3 deletions in the context of clinical treatment. This feature of the WHO protocol is intentional because it captures clinically important pfhrp2/3 deletions and enables real-time, efficient signalling to malaria control programmes of a potential problem. However, it also introduces selection bias that requires careful consideration when estimating the true prevalence of pfhrp2/3-deleted parasites. We overcame this limitation by using a conservative  1344600  1344874  1345220  1345600  1353662  1353972  1354234  1354476  1358322  1358598  1358968  1359538  1360112  1360530  1361034  1361356  1361784  1362373  1362769  1363090  1363486  1365604  1365932  1366208  1366440  1366699  1366996  1367252  1367511  1371899  1372268  1372522  HRP2_1  HRP2_2  HRP2_3  HRP2_4  HRP2_5  HRP2_6  HRP2_7  HRP2_8  1383867  1384136  1384462  1386662  1386880  1387124  1387380  1392344  1392760  approach that incorporated both RDT and PCR data to estimate false-negative RDT results due to pfhrp2 deletions. This metric is relevant to control programmes, but does not capture asymptomatic or low parasite-density infections by pfhrp2/3-deleted parasites.
Second, only a subset of samples underwent advanced analysis to expedite reporting, and clinical data were not available for all participants. This was not unexpected for a pragmatic field study of this size. We do not believe that it introduced sufficient bias into  1325735  1327735  1329735  1331735  1333735  1335735  1337735  1339735  1341735  1343735  1345735  1347735  1349735  1351735  1353735  1355735  1357735  1359735  1361735  1363735  1365735  1367735  1369735  1371735  1373735  1375735  1377735  1379735  1381735  1383735  1385735  1387735  1389735  1391735  1393735  1395735  1397735  1399735  1401735  1403735  1405735  1407735  1409735  1411735  1413735  1415735  1417735  1419735  1421735  our prevalence estimates or population genetic analysis to change our conclusions. Third, we cannot comment on changes in selection pressure over time because the study was cross-sectional. Fourth, we only sampled three regions of Ethiopia, which is a diverse and populous country. The Federal Ministry of Health is now conducting a country-wide survey that will enable comparison of pfhrp2/3 deletions over time in select sites. Our large prospective study, using established molecular and antigen detection methods, and a targeted sequencing approach show that phrp2/3-deleted P. falciparum is a common cause of false-negative RDT results in two regions of Ethiopia. Concerningly, these genomic tools reveal evidence of recent, strong selection for pfhrp2 deletion in the regions sampled. The selective pressures favouring pfhrp2-deleted parasites appear to have occurred on a background of pre-existing pfhrp3 deletions. Existing malaria control programmes in the region are threatened by expansion of these parasite strains, and prevalence estimates in Tigray and Amhara exceed WHO-recommended thresholds for RDT change. Surveillance has already informed decisions to deploy alternative malaria diagnostics in Eritrea and Djibouti and is underway in Somalia and Sudan. Urgent attention to these deletion mutations is needed to inform malaria diagnostic testing policies throughout the Horn of Africa.

Methods
Study design and data collection. We performed a cross-sectional, multisite study in 11 districts along Ethiopia's borders with Eritrea, Sudan and South Sudan, located within three of its nine administrative regions. On average, ten health facilities were selected from each district, including four districts of Amhara Region (northwest Ethiopia), six districts of Tigray Region (north Ethiopia) and one district of Gambella region (southwest Ethiopia) during the 2017-2018 peak malaria transmission season (September-December, although enrolment in Gambella was completed in April 2018) (Fig. 1). Per WHO protocol 30 , each facility passively enroled participants presenting with symptoms of malaria (fever, headache, joint pain, feeling cold, nausea and/or poor appetite), with sample size proportionally allocated to each facility based on the previous year's malaria case load. All participants provided informed consent, participated in interview questionnaires, underwent blood collection for RDT testing using two types of RDT and were treated according to national guidelines. Data were double-entered into Epi Info (v.3.2), and discrepancies resolved using original paper forms by consensus. Ethical approval was obtained from the Ethiopia Public Health Institute (EPHI) Institutional Review Board (IRB; protocol EPHI-IRB-033-2017) and WHO Research Ethics Review Committee (protocol ERC.0003174 001). Processing of de-identified samples and data at the University of North Carolina at Chapel Hill (UNC) was determined to constitute non-human subjects research by the UNC IRB (study 17-0155). The study was determined to be non-research by the Centers for Disease Control (CDC) and Prevention Human Subjects office (0900f3eb81bb60b9). Experiments were performed in accordance with relevant guidelines and regulations.

Field sample evaluation. Study participants were evaluated using both a CareStart
Pf/Pv (HRP2/Pv-pDH) RDT (Access Bio, catalogue no. RM VM-02571) and an SD Bioline Malaria Ag P.f. (HRP2/Pf-LDH) RDT (Alere, catalogue no. 05FK90). For the CareStart RDT, 5 µl of capillary whole blood was collected by fingerprick and transferred to the RDT sample well, along with 60 µl of buffer solution. Results were read at 20 min. The SD Bioline RDT followed the same protocol, but with four drops of buffer added and the results read in a 15-30 min window. Participants testing positive by either RDT were first prescribed treatment, according to Ethiopian national guidelines 50 .
Cases with any positive HRP2 or Pf-LDH RDT band were considered positive for P. falciparum malaria. Cases that were Pf-LDH-positive but HRP2-negative on both RDTs were considered potential candidates for pfhrp2/3 gene deletion and defined as 'discordant' . These participants, along with a subset of HRP2-positive and HRP2-negative controls, provided further informed consent for additional blood collection for dried blood spot (DBS) preparation. At least two DBS samples ( 55 to create high-quality consensus sequences from sequence read data utilizing unique molecular indexes (UMIs) of MIPs, maps those sequences to the reference genome using bwa (v.0.7.17) and removes off-target sequences as described previously 33,54 . Deletion calls were limited to samples that had high coverage to avoid false positives. Considering the high frequency of large deletions present in the sample set, the coverage threshold was based on a subset of probes that were present on >60% of the samples, none of which overlapped with the chromosome 8 or 13 deletions. Samples with a median coverage of fewer than five UMIs for this subset of probes were excluded from analysis. Structural profiling was performed using the UMI count table (Supplementary Table 7). The count table was converted to a presence/absence table such that if a probe had more than one UMI for a given sample, it was accepted as present (that is, not deleted). Samples were clustered into subtelomeric structural profile groups based on this table using the hierarchical clustering algorithm AgglomerativeClustering of the Python module Scikit-learn (v.0.20) 56  Initial variant calls were made using freebayes (v1.3.1) via MIPTools with the following options:--pooled-continuous--min-base-quality 1--min-alternate-fraction 0.01--min-alternate-count 2--haplotype-length -1--min-alternate-total 10--use-best-n-alleles 70--genotype-qualities. Variants were processed using MIPTools to filter for: variant quality >1, genotype quality >1, average alternate allele quality >15, minimum depth >2 UMIs; and make final genotype calls based on the major allele (within-sample allele frequency >0.5). In addition, the following variants were removed from the final call set: those that were observed as a major allele in less than two samples (singletons), not supported by more than two UMIs in at least three samples, present on multicopy genes, and indels. Variant calls were further filtered for missingness to avoid imputation in EHH calculations: samples missing calls for >50% of the variants were removed, variants missing calls in >50% of the samples were removed. Variants calls were converted to.map and.hap files (Supplementary Table 8) for use with the rehh package (v3.1.2) in R.

Assessment of MIP calls using whole-genome sequencing.
We performed WGS on a subset of samples selected by convenience to assess the accuracy of MIP pfhrp2/3 deletion calls. DNA extracted from samples with discordant RDT results were selected for P. falciparum selective whole-genome amplification (sWGA) and whole-genome sequencing exactly as described previously 31 . In brief, DNA was first subjected to two separate sWGA reactions using the Probe_10 primer set described by Oyola et al 57 and the JP9 primer set 31 . sWGA products were then pooled in equal volumes and acoustically sheared using a Covaris E220 instrument before to sequencing library preparation using Kappa Hyper library preps (Roche, catalogue no. KK8504). Indexed libraries were then pooled and sequenced on an Illumina HiSeq 4000 instrument using 150 bp, paired-end sequencing. Sequencing reads were deposited into NCBI's Sequence Read Archive (PRJNA742125).
Published whole-genome sequencing data retrieval. Fastq files from 25 Ethiopian samples included in the MalariaGEN genome variation project 19 and three laboratory strains (3D7, HB3 and DD2) from MalariaGEN genetic crosses project 58 were downloaded from the European Nucleotide Archive using fasterq-dump (v.2.10.8) and sample accession numbers on 19 September 2020 (Supplementary Table 9).
Variants were filtered for: variant quality >20, genotype quality >15, average alternate allele quality >15, minimum depth >4 reads. In addition, the following variants were removed from the final call set: those that were never observed and saponin (MilliporeSigma, catalogue no. 47036-250G-F) as described previously 51 . Quantitative PCR (qPCR) assays were first performed in duplicate for the P. falciparum lactate dehydrogenase gene (pfldh) 52 . To avoid the risk of misclassification due to DNA concentrations below the limit of detection for pfhrp2/3 PCR assays, further analysis was restricted to samples with >100 parasites per µl by qPCR (Extended Data Fig. 1) 13 . PCR assays targeting exon 2 of pfhrp2 and pfhrp3 were then performed in duplicate as described previously 6 , except that PCR reactions were performed as single-step, 45-cycle assays, using 10 µl of template and AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, catalogue no. 4398813) in a 25 µl reaction volume. In addition to no-template and P. falciparum 3D7 strain (pfhrp2+/3+) positive controls, pfhrp2 assays included an additional DD2 strain (pfhrp2−/3+) control and pfhrp3 assays included an additional HB3 strain (pfhrp2+/3−) control. Finally, an additional single-copy gene, real-time PCR assay targeting P. falciparum β-tubulin was performed to confirm that sufficient parasite DNA remained in samples with a negative pfhrp2/3 PCR result 13 . Pfhrp2/3 genotyping calls were made in samples with pfldh qPCR parasitaemia >100 parasites per µl to avoid misclassification in the setting of amplification failure due to low target DNA concentration. A pfhrp2 or pfhrp3 positive call required one or more replicate with distinct band(s) with the expected fragment length. A negative call required both pfhrp2 or pfhrp3 replicates to be negative. Detailed reaction conditions for all PCR assays are described in the Supplementary File. Serological assays. The presence of HRP2, pan-LDH and aldolase antigenaemia was assessed in a subset of DBS samples (single 6 mm punch) using a multiplex bead-based immunoassay exactly as described previously 12 . Within this multiplex assay, capture and detection antibodies against the HRP2 antigen would also recognize similar epitopes on the HRP3 antigen, so unique signals for these two antigens cannot be obtained.
Prevalence estimates. We estimated the prevalence of P. falciparum infections expected to have false-negative HRP2-based RDT results due to pfhrp2 deletions as follows. First, we calculated the proportion of all RDT-positive P. falciparum cases (HRP2+ or Pf-LDH+ on any RDT) with the discordant RDT profile (HRP2− on both RDTs, but Pf-LDH+), overall and by region. Second, we calculated the observed concordance between the discordant RDT profile and a pfhrp2-negative PCR call, overall. Prevalence estimates and 95% CIs were then back-transformed overall and by region using the ci.impt function within the asbio R package (v.1.5-5), which generates CI values for the product of two proportions using delta derivation. This allowed us to estimate with confidence the proportion of P. falciparum infections with both pfhrp2 deletions and false-negative HRP2-based RDT results, overall and by region. As a sensitivity analysis, we also estimated the proportion of those with a discordant RDT and a pfhrp2-negative PCR call (directly multiplying the true proportion of P. falciparum-positive individuals with a discordant RDT profile, overall and by region, by 0.727, or the overall proportion of discordant RDT samples that had a pfhrp2− PCR result). The 95% CI values were then generated using bootstrapping (1,000 iterations). The prevalence estimates and CI values generated by the two approaches were similar (Supplementary Table 3).

Pfhrp2/3 molecular inversion probe (MIP) development.
Pfhrp2, pfhrp3 and the flanking regions within a 100 kb window surrounding each gene were targeted for MIP designs using MIPTools 53 . A tiled design strategy was employed that involved multiple, overlapping probes spanning each gene target. Twenty-two genes flanking pfhrp2 and 31 genes flanking pfhrp3 were used in the design, of which 11 and 19 were successful on the first design try, respectively. A second attempt was not made for designs for the flanking genes. A total of 241 probes were designed: 9 for pfhrp2, 9 for pfhrp3 and 223 for the flanking genes. MIPs were designed using the 3D7 (v.3) reference genome avoiding hybridization arms in variant regions when possible. Eighty alternative probes accommodating potential variants in the highly variable pfhrp2 and pfhrp3 genes were also created. A 15.5 kb segment centromeric to pfhrp3 on chromosome 13 between positions 2,792,000 and 2,807,500 is duplicated on chromosome 11 between positions 1,918,007 and 1,933,488, with 99.4% sequence identity. Therefore, the target genes falling into this region were multicopy genes and their probes were designed to bind to both loci on the genome (see Supplementary Table 5 for the design overview including all genes targeted, MIPs designed and genomic coordinates). Probes were ordered from Integrated DNA Technologies as 200 pmol ultramer oligos. Probe sequences are provided in the Supplementary Table 6.
MIP capture and deep sequencing of clinical samples. All DNA samples extracted by UNC underwent MIP capture using the capture and amplification methods exactly as described by Verity et al. 54 , with the exception of oligonucleotides (the pfhrp2/3 MIP oligonucleotide panel described above was used) and controls (we selected a different set of controls that are informative for pfhrp2/3 deletion characterization). All MIP captures included multiple controls: 3D7 (pfhrp2+/3+), DD2 (pfhrp2−/3+), HB3 (pfhrp2+/3−) laboratory strains; as well as low-and high concentration mixes (1% HB3, 10% DD2, 89% 3D7) at densities of 250 and 1,000 parasites per µl, respectively. Samples were sequenced on the Illumina NextSeq 550 instrument using 150 bp paired-end sequencing and dual indexing. as a major allele in any sample, not supported by more than ten reads in at least one sample, and indels. Final genotype calls were based on the major allele (within-sample allele frequency >0.5). Variant calls were further filtered for missingness to avoid imputation in EHH calculations: samples missing calls for >95% of the variants were removed, variants missing calls in >10% of the samples were removed.
Telomeric profiling of the published genomes was carried out by visual inspection of depth-of-coverage plots (Extended Data Figs. 8 and 9). Summary statistics were generated (Supplementary Table 10) using the Python pandas module (v.0.23).
Statistical and population genetic analysis. Data collected during the participant's study visit (clinical data and RDT results) were linked to laboratory results via the barcode number transcribed on DBS sent to the UNC and CDC laboratories. Samples in the dataset with missing or duplicate barcodes were arbitrated using original paper questionnaires by the EPHI data centre. An analysis dataset that included both PCR and field data was created including all samples that we could confidently merge by both barcode number and region label.
EHH statistics were calculated to evaluate the regions flanking the pfhrp2 and pfhrp3 genes for signatures of recent positive selection 36 using the rehh package (v.3.1.2) 59 . EHH statistics were calculated using the data2haplohh and calc_ehh functions, haplotype furcations were calculated using calc_furcation, and plots were generated using the package's plot function and annotated using Inkscape (v.0.92).
Statistics and reproducibility. Sample sizes were chosen based on the WHO protocol 30 . Sources of data and samples included in the study are outlined in Fig. 2. DBS samples were only collected from a subset of participants based on the WHO protocol. Molecular, immunological and sequencing assays were performed on random subsets selected by EPHI. Most analyses were limited to samples that could be matched unambiguously across datasets. For example, any DBS samples found to have identical participant IDs were excluded from analysis. Similarly, DBS labelled with a participant and region ID that did not match clinical data were excluded from most analyses. These accounted for a minority of participants. Discordances in participant IDs and DBS sample labels were resolved whenever possible.
Field staff were not blinded to malaria RDT results because they were used to inform clinical care according to national guidelines. Pfhrp2/3 deletion calls using MIP sequencing were made by an investigator who was blinded to clinical data (including RDT results), HRP2 immunoassay results and pfhrp2/3 deletion calls using PCR.

Software and code
Policy information about availability of computer code Data collection Data was collected in the field using paper forms and double entered into Epi Info (v3.2). Discrepancies were cross-checked against the hardcopy paper forms and resolved by consensus.

Data analysis
Custom code used during data analysis is available through GitHub at https://doi.org/10.5281/zenodo.5160363. Statistical analysis was performed using R (version 3.6.0, R Core Team, Vienna, Austria, 2019; www.R-project.org). Prevalence estimates were generated using the asbio package (v1. . 95% confidence intervals were calculated using the binom.confint package (v1. Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability MIP and genomic sequencing data is available through the Sequence Read Archive (PRJNA742125). De-identified datasets generated during the current study and used to make all figures are available as supplementary files or tables.
Extended Data Figure 6-7 were derived from genomic sequencing data made publicly available by MalariaGEN (https://www.malariagen.net/data, downloaded Sep 19 2020). Extended Data Figure 8 was derived from genomic sequencing data generated during this study and publicly available through MalariaGEN.
The hg38 human genome used during whole-genome sequencing analysis was downloaded from the US National Institutes of Health National Center for Biotechnology and Information database on December 2, 2015 (available at https://www.ncbi.nlm.nih.gov/assembly/GCF_000001405.26/).

Field-specific reporting
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Life sciences study design
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Sample size
Sample sizes were derived from the WHO "Template protocols to support surveillance and research for pfhrp2/pfhrp3 gene deletions," available at https://www.who.int/malaria/publications/atoz/hrp2-deletion-protocol/en/.
Data exclusions Sources of data and samples included in the study are outlined in Figure 2. Dried blood spot samples were only collected from a subset of subjects based on WHO protocols. Molecular, immunological, and sequencing assays were performed on random subsets selected by EPHI. As outlined in the Methods, most analyses were limited to samples that could be matched unambiguously across datasets. For example, any DBS samples found to have identical participant IDs were excluded from analysis. Similarly, DBS labeled with a participant and region ID that did not match clinical data were excluded from most analyses. These accounted for a minority of subjects. Discordances in participant IDs and DBS sample labels were resolved whenever possible.

Replication
All PCR assays were performed in duplicate. Deletion calls made by PCR were limited to samples with >100 parasites/μL, with negative pfhrp2 or pfhrp3 bands in both replicates, and positive by a final confirmatory real-time PCR assay as described in the Methods.
Comparison of whole-genome sequencing and MIP calls was undertaken for 14 samples as outlined in the Results. Sequencing (MIP and WGS) was performed across multiple flow cells.
To increase confidence in pfhrp2/3 deletion calls, multiple confirmatory methods were employed, including PCR, MIP sequencing, WGS, and an HRP2 immunoassay.
Results were compared across platforms, and concordance/discordance between methods included in the Results. While most calls were concordant, we did observe samples with discordance results across different assays. This was not unexpected because the assay targets are different in some cases (ex: HRP2 immunoassay detects HRP2 or HRP3 antigen that can linger after clearance of parasitemia, whereas the molecular methods detect parasite DNA that clears rapidly after resolution of infection). However, we cannot exclude the possibility that some discordance may have been introduced by ambiguous sample labeling and/or processing during the conduct of the field work. We overcame this by restricting analyses to samples with complete meta-data and no ambiguity when merged with molecular and/or antigen data (see Figure 2), and by employing a conservative approach to prevalence estimates as described in the Results, Methods, and Discussion.
Randomization As outlined in the WHO protocol, any subject presenting to study health facilities with symptoms of malaria was eligible for enrollment.
Randomization was not performed. We did not undertake detailed analyses of covariates, except as shown in Supplementary Table 1 in which we stratified by pfhrp2/3 status.

Blinding
Field staff were not blinded to malaria RDT results because they were used to inform clinical care according to national guidelines.
Pfhrp2/3 deletion calls using MIP sequencing were made by an investigator who was blinded to clinical data (including RDT results), HRP2 immunoassay results, and pfhrp2/3 deletion calls using PCR.
Reporting for specific materials, systems and methods