False-negative Rapid Diagnostic Tests Results Research Articles

Imported malaria into Australia: surveillance insights and opportunities.

Malaria continues to pose a significant burden in endemic countries, many of which lack access to molecular surveillance. Insights from malaria cases in travellers returning to non-endemic areas can provide valuable data to inform endemic country programmes. To evaluate the potential for novel global insights into malaria, we examined epidemiological and molecular data from imported malaria cases to Australia. We analysed malaria cases reported in Australia from 2012 to 2022 using National Notifiable Disease Surveillance System data. Molecular data on imported malaria cases were obtained from literature searches. Between 2012 and 2022, 3204 malaria cases were reported in Australia. Most cases (69%) were male and 44% occurred in young adults aged 20-39years. Incidence rates initially declined between 2012 and 2015, then increased until 2019. During 2012-2019, the incidence in travellers ranged from 1.34 to 7.71 per 100 000 trips. Cases were primarily acquired in Sub-Saharan Africa (n = 1433; 45%), Oceania (n = 569; 18%) and Southern and Central Asia (n = 367; 12%). The most common countries of acquisition were Papua New Guinea (n = 474) and India (n = 277). Plasmodium falciparum accounted for 58% (1871/3204) of cases and was predominantly acquired in Sub-Saharan Africa, and Plasmodium vivax accounted for 32% (1016/3204), predominantly from Oceania and Asia. Molecular studies of imported malaria cases to Australia identified genetic mutations and deletions associated with drug resistance and false-negative rapid diagnostic test results, and led to the establishment of reference genomes for P. vivax and Plasmodium malariae. Our analysis highlights the continuing burden of imported malaria into Australia. Molecular studies have offered valuable insights into drug resistance and diagnostic limitations, and established reference genomes. Integrating molecular data into national surveillance systems could provide important infectious disease intelligence to optimize treatment guidelines for returning travellers and support endemic country surveillance programmes.

Molecular investigation of malaria-infected patients in Djibouti city (2018–2021)

BackgroundThe Republic of Djibouti is a malaria endemic country that was in pre-elimination phase in 2006–2012. From 2013, however, malaria has re-emerged in the country, and its prevalence has been increasing every year. Given the co-circulation of several infectious agents in the country, the assessment of malaria infection based on microscopy or histidine-rich protein 2 (HRP2)-based rapid diagnostic tests (RDT) has shown its limitations. This study, therefore, aimed to assess the prevalence of malaria among febrile patients in Djibouti city using more robust molecular tools.MethodsAll suspected malaria cases reported to be microscopy-positive were randomly sampled (n = 1113) and included in four health structures in Djibouti city over a 4-year period (2018–2021), mainly during the malaria transmission season (January–May). Socio-demographic information was collected, and RDT was performed in most of the included patients. The diagnosis was confirmed by species-specific nested polymerase chain reaction (PCR). Data were analysed using Fisher’s exact test and kappa statistics.ResultsIn total, 1113 patients with suspected malaria and available blood samples were included. PCR confirmed that 788/1113 (70.8%) were positive for malaria. Among PCR-positive samples, 656 (83.2%) were due to Plasmodium falciparum, 88 (11.2%) Plasmodium vivax, and 44 (5.6%) P. falciparum/P. vivax mixed infections. In 2020, P. falciparum infections were confirmed by PCR in 50% (144/288) of negative RDTs. After the change of RDT in 2021, this percentage decreased to 17%. False negative RDT results were found more frequently (P < 0.05) in four districts of Djibouti city (Balbala, Quartier 7, Quartier 6, and Arhiba). Malaria occurred less frequently in regular bed net users than in non-users (odds ratio [OR]: 0.62, 95% confidence interval [CI]: 0.42–0.92).ConclusionsThe present study confirmed the high prevalence of falciparum malaria and, to a lesser extent, vivax malaria. Nevertheless, 29% of suspected malaria cases were misdiagnosed by microscopy and/or RDT. There is a need to strengthen the capacity for diagnosis by microscopy and to evaluate the possible role of P. falciparum hrp2 gene deletion, which leads to false negative cases of P. falciparum.

Surveillance of pfhrp2 and pfhrp3 gene deletions among symptomatic Plasmodium falciparum malaria patients in Central Vietnam

BackgroundMalaria rapid diagnostic tests (RDTs) remain the main point-of-care tests for diagnosis of symptomatic Plasmodium falciparum malaria in endemic areas. However, parasites with gene deletions in the most common RDT target, histidine rich protein 2 (pfhrp2/HRP2), can produce false-negative RDT results leading to inadequate case management. The objective of this study was to determine the prevalence of hrp2/3 deletions causing false-negative RDT results in Vietnam (Gia Lai and Dak Lak provinces).MethodsIndividuals presenting with malaria symptoms at health facilities were screened for P. falciparum infection using light microscopy and HRP2-RDT (SD Bioline Malaria Antigen Pf/Pv RDT, Abbott). Microscopically confirmed P. falciparum infections were analysed for parasite species by 18S rRNA qPCR, and pfhrp2 and pfhrp3 exon2 deletions were investigated by nested PCR. pfhrp2 amplicons were sequenced by the Sanger method and HRP2 plasma levels were determined by enzyme-linked immunosorbent assay (ELISA).ResultsThe prevalence of false-negative RDT results among symptomatic cases was 5.6% (15/270). No pfhrp2 and pfhrp3 deletions were identified. False-negative RDT results were associated with lower parasite density (p = 0.005) and lower HRP2 plasma concentrations (p < 0.001), as compared to positive RDT.ConclusionsThe absence of hrp2/3 deletions detected in this survey suggests that HRP2-based malaria RDTs remain effective for the diagnosis of symptomatic P. falciparum malaria in Central Vietnam.

Plasmodium falciparum histidine-rich protein 2 and 3 gene deletion in the Mount Cameroon region

ObjectivePlasmodium falciparum produces histidine-rich protein 2/3 (Pfhrp2/3) genes that accumulate to high levels in the bloodstream and serve as a diagnostic and prognostic marker for falciparum malaria. Pfhrp2/3 gene deletions may lead to false-negative rapid diagnostic test (RDT) results. We aimed to determine the prevalence of pfhrp2/3 gene deletions in P. falciparum isolates and the implications for RDT use in the Mount Cameroon region. MethodsA cross-sectional hospital-based study with malaria diagnosis performed using microscopy, RDT and nested polymerase chain reaction (nPCR). In total, 324 P. falciparum microscopy positive individuals were enrolled and their samples confirmed positive for P. falciparum using 18SrRNA PCR. Samples that gave false-negative RDT results were analyzed to detect pfhrp2/3 exon 2 deletions. ResultsOf 324 positive microscopic and nPCR samples, 16 gave RDT false-negative results. Among the 324 P. falciparum positive isolates, exon 2 deletions were observed in 2.2% (7 of 324); 3 were negative for pfhrp2 gene, 2 for pfhrp3, and 2 for both pfhrp2 and pfhrp3 (double deletions). ConclusionP. falciparum isolates with pfhrp2/3 gene deletion were present in the parasite populations and may contribute to the RDT false-negative results in the Mount Cameroon region.

Prevalence of Pfhrp2 and Pfhrp3 Gene Deletions in Plasmodium Falciparum Isolates and their Performance of Hrp2 Based Malaria Rapid Diagnostic Tests in Three Districts of Ghana

Malaria rapid diagnostic tests (MRDTs) are important for malaria disease management. However, the performance of the RDTs is affected when the targeted antigens in the parasite have a variation or are altogether absent. The most common parasite target antigen in RDTs, Plasmodium falciparum histidine-rich protein 2 (HRP2), has been reported to be absent in some P. falciparum parasites. 371 patient samples, from Akuapem North (58.5%), Atiwa East (21.3%), and from New Juaben (20.2%), were used in the study. PCR provided the highest number, 14.8% (55/371), of positive detections for falciparum infections. Microscopy detected parasites in 20/261 (7.7%) samples, and the minimum parasite density by microscopy was 430 parasites/µL. Out of the 371 samples, 27 (7.3%) were positive by RDT. The highest RDT positivity rate, 13.3% (10/75), was observed at New Juaben. False-negative RDT results were obtained in 43/55 (78.2%) of the negative branded RDT kits. Only two microscopies positive sample were RDT positive. Using 18SrDNA PCR, 55 (14.8%) samples were positive for P. falciparum. In Akuapem North, 79.2 % (19/24) of the PCR positive samples had P. falciparum parasites that lacked exon 2 of PFHRP2. An overall RDT positivity rate of 7.3% (27/371) and false-negative rate of 78.2% (43/55) were observed for the study sites. Plasmodium falciparum parasite populations with deletions of the PFHRP2 and PFHRP3 genes are present in Ghana. There is an urgent need to investigate the prevalence and geographic distribution of these parasites. Keywords: Histidine Rich Protien (HRP), Malaria Rapid Diagnostic Test (MRDT), Malaria, Rapid Diagnostic Test (RDT).

No evidence of false-negative Plasmodium falciparum rapid diagnostic results in Monrovia, Liberia

BackgroundMalaria diagnosis in many malaria-endemic countries relies mainly on the use of rapid diagnostic tests (RDTs). The majority of commercial RDTs used in Africa detect the Plasmodium falciparum histidine-rich protein 2 (PfHRP2). pfhrp2/3 gene deletions can therefore lead to false-negative RDT results. This study aimed to evaluate the frequency of PCR-confirmed, false-negative P. falciparum RDT results in Monrovia, Liberia.MethodsPfHRP2-based RDT (Paracheck Pf®) and microscopy results from 1038 individuals with fever or history of fever (n = 951) and pregnant women at first antenatal care (ANC) visit (n = 87) enrolled in the Saint Joseph’s Catholic Hospital (Monrovia) from March to July 2019 were used to assess the frequency of false-negative RDT results. True–false negatives were confirmed by detecting the presence of P. falciparum DNA by quantitative PCR in samples from individuals with discrepant RDT and microscopy results. Samples that were positive by 18S rRNA qPCR but negative by PfHRP2-RDT were subjected to multiplex qPCR assay for detection of pfhrp2 and pfhrp3.ResultsOne-hundred and eighty-six (19.6%) and 200 (21.0%) of the 951 febrile participants had a P. falciparum-positive result by RDT and microscopy, respectively. Positivity rate increased with age and the reporting of joint pain, chills and shivers, vomiting and weakness, and decreased with the presence of coughs and nausea. The positivity rate at first ANC visit was 5.7% (n = 5) and 8% (n = 7) by RDT and microscopy, respectively. Out of 207 Plasmodium infections detected by microscopy, 22 (11%) were negative by RDT. qPCR confirmed absence of P. falciparum DNA in the 16 RDT-negative but microscopy-positive samples which were available for molecular testing. Among the 14 samples that were positive by qPCR but negative by RDT and microscopy, 3 only amplified pfldh, and among these 3 all were positive for pfhrp2 and pfhrp3.ConclusionThere is no qPCR-confirmed evidence of false-negative RDT results due to pfhrp2/pfhrp3 deletions in this study conducted in Monrovia (Liberia). This indicates that these deletions are not expected to affect the performance of PfHRP2-based RDTs for the diagnosis of malaria in Liberia. Nevertheless, active surveillance for the emergence of PfHRP2 deletions is required.

Rapid diagnostic test negative Plasmodium falciparum malaria in a traveller returning from Ethiopia

BackgroundPlasmodium falciparum strains with mutations/deletions of the genes encoding the histidine-rich proteins 2/3 (pfhrp2/3) have emerged during the last 10 years leading to false-negative results in HRP2-based rapid diagnostic tests (RDTs). This can lead to unrecognized infections in individuals and to setbacks in malaria control in endemic countries where RDTs are the backbone of malaria diagnostics and control.Case descriptionHere the detection of a pfhrp2/3-negative P. falciparum infection acquired in Ethiopia by a 63-year old female traveller is presented. After onset of symptoms during travel, she was first tested negative for malaria, most probably by RDT, at a local hospital in Harar, Ethiopia. Falciparum malaria was finally diagnosed microscopically upon her return to Germany, over 4 weeks after infection. At a parasite density of approximately 5387 parasites/µl, two different high-quality RDTs: Palutop + 4 OPTIMA, NADALRMalaria PF/pan Ag 4 Species, did not respond at their respective P. falciparum test lines. pfhrp2/3 deletion was confirmed by multiplex-PCR. The patient recovered after a complete course of atovaquone and proguanil. According to the travel route, malaria was acquired most likely in the Awash region, Central Ethiopia. This is the first case of imported P. falciparum with confirmed pfhrp2/3 deletion from Ethiopia.ConclusionHRP2-negative P. falciparum strains may not be recognized by the presently available HRP2-based RDTs. When malaria is suspected, confirmation by microscopy and/or qPCR is necessary in order to detect falciparum malaria, which requires immediate treatment. This case of imported P. falciparum, non-reactive to HRP2-based RDT, possibly underlines the necessity for standardized, nationwide investigations in Ethiopia and should alert clinicians from non-endemic countries to the possibility of false-negative RDT results which may increase in returning travellers with potentially life-threatening infections.

Diagnostic performance of HRP2-only malaria rapid diagnostic test in Ghanaian pregnant women with asymptomatic peripheral blood infection: the case of First Response® test kit

Background: The use of malaria rapid diagnostic test(RDT) enables targeted treatment that mitigates against the development of parasite drug resistance. With detection thresholds at ≥200 parasites/μL, their diagnostic performance in pregnant women may be challenging as asymptomatic infections with low parasite densities are common. Few data exist on the performance of histidine-rich protein-2 (HRP2) RDTs in Ghanaian pregnant women considering commonly occurring low parasite densities. Objective: The study sought to contribute more knowledge on test performance on First Response® test kit to guide the continuous evaluation of HRP2 RDTs in pregnancy. Methods: As part of an antimalarial drug trial in pregnancy, First Response® RDT results were compared to microscopy of peripheral blood slides in 1664 women. The diagnostic performance indicators were computed as proportions with 95% confidence intervals. The risk of having a positive RDT result was computed for age, gravidity and parasite density using binomial regression methods. Results: Parasitaemia prevalence by microscopy was 5.71%(n =95/1664) while that by RDT was 21.57%(n = 359/1664). Sensitivity was 82.11% (n=78/95), specificity was 82.09% (n=1288/1569), positive predictive value was 21.73% (n=78/359), and the likelihood ratio for a positive test was 4.58. False-negative RDT results were recorded for low parasite densities as well as densities ≥ 200/ μL while false-positive results were recorded in 281of 1664 tests. Primigravidae and younger aged women were more likely to have positive RDT results compared to multigravidae and women aged ≥ 30 yr. Conclusion: The moderate sensitivity, specificity and other diagnostic parameters reported suggest the First Response® malaria RDT is useful for detecting peripheral parasitaemia in pregnant women but the use of HRP2-only RDTs is limited by the existence of parasites with HRP2 gene deletion. The use of RDTs based on combined antigens continues to be recommended. Further research is needed on RDT performance in pregnant women with declining malaria transmission.

Lineage-Specific Expansion of Plasmodium falciparum Parasites With pfhrp2 Deletion in the Greater Mekong Subregion.

Deletion of the pfhrp2 gene in Plasmodium falciparum can lead to false-negative rapid diagnostic test (RDT) results, constituting a major challenge for evidence-based malaria treatment. Here we analyzed the whole genome sequences of 138 P. falciparum clinical samples collected from the China-Myanmar boarder for pfhrp2 and pfhrp3 gene deletions. We found pfhrp2 and pfhrp3 deletions in 9.4% and 3.6% of samples, respectively, with no samples harboring deletions of both genes. The pfhrp2 deletions showed 2 distinct breakpoints, representing 2 different chromosomal deletion events. A phylogenetic analysis performed using genome-wide single-nucleotide polymorphisms revealed that the 2 pfhrp2 breakpoint groups as well as all the pfhrp3-negative parasites formed separate clades, suggesting they might have resulted from clonal expansion of pfhrp2- and pfhrp3-negative parasites. These findings highlight the need for urgent surveys to determine the prevalence of pfhrp2-negative parasites causing false-negative RDT results and a plan for switching of RDTs pending the survey results.

Collaborative intelligence and gamification for on-line malaria species differentiation

BackgroundCurrent World Health Organization recommendations for the management of malaria include the need for a parasitological confirmation prior to triggering appropriate treatment. The use of rapid diagnostic tests (RDTs) for malaria has contributed to a better infection recognition and a more targeted treatment. Nevertheless, low-density infections and parasites that fail to produce HRP2 can cause false-negative RDT results. Microscopy has traditionally been the methodology most commonly used to quantify malaria and characterize the infecting species, but the wider use of this technique remains challenging, as it requires trained personnel and processing capacity.ObjectiveIn this study, the feasibility of an on-line system for remote malaria species identification and differentiation has been investigated by crowdsourcing the analysis of digitalized infected thin blood smears by non-expert observers using a mobile app.MethodsAn on-line videogame in which players learned how to differentiate the young trophozoite stage of the five Plasmodium species has been designed. Images were digitalized with a smartphone camera adapted to the ocular of a conventional light microscope. Images from infected red blood cells were cropped and puzzled into an on-line game. During the game, players had to decide the malaria species (Plasmodium falciparum, Plasmodium malariae, Plasmodium vivax, Plasmodium ovale, Plasmodium knowlesi) of the infected cells that were shown in the screen. After 2 months, each player’s decisions were analysed individually and collectively.ResultsOn-line volunteers playing the game made more than 500,000 assessments for species differentiation. Statistically, when the choice of several players was combined (n > 25), they were able to significantly discriminate Plasmodium species, reaching a level of accuracy of 99% for all species combinations, except for P. knowlesi (80%). Non-expert decisions on which Plasmodium species was shown in the screen were made in less than 3 s.ConclusionThese findings show that it is possible to train malaria-naïve non-experts to identify and differentiate malaria species in digitalized thin blood samples. Although the accuracy of a single player is not perfect, the combination of the responses of multiple casual gamers can achieve an accuracy that is within the range of the diagnostic accuracy made by a trained microscopist.

Implications of Parasites Lacking Plasmodium falciparum Histidine-Rich Protein 2 on Malaria Morbidity and Control When Rapid Diagnostic Tests Are Used for Diagnosis.

Rapid diagnostic tests (RDTs) are an important tool for malaria diagnosis, with most using antibodies against Plasmodium falciparum histidine-rich protein 2 (PfHRP2). Reports of P. falciparum lacking this protein are increasing, creating a problem for diagnosis of falciparum malaria in locations without quality-assured microscopy. An agent-based stochastic simulation model of P. falciparum transmission was used to investigate the selective pressure exerted on parasite populations by use of RDTs for diagnosis of symptomatic cases. The model considered parasites with normal, reduced, or no PfHRP2, and diagnosis using PfHRP2-only or combination RDTs. Use of PfHRP2-only RDTs in communities where a PfHRP2-negative parasite was introduced during the simulation resulted in transmission of the parasite in >80% of cases, compared with <30% for normal or PfHRP2-reduced parasites. Using PfHRP2-only RDTs in the presence of PfHRP2-negative parasites caused an increase in prevalence, reduced RDT positivity within symptomatic patients but no change in the number of antimalarial treatments due to false-negative RDT results. Diagnosis with PfHRP2/Pf-Plasmodium lactate dehydrogenase combination RDTs did not select for PfHRP2-negative parasites. The use of PfHRP2-only RDTs is sufficient to select P. falciparum parasites lacking this protein, thus posing a significant public health problem, which could be moderated by using PfHRP2/Pf-Plasmodium lactate dehydrogenase combination RDTs.

Rapid diagnostic tests failing to detect Plasmodium falciparum infections in Eritrea: an investigation of reported false negative RDT results

BackgroundRelatively large number of false-negative malaria rapid diagnostic test (RDT) results for microscopically confirmed Plasmodium falciparum cases were reported from five of the six administrative regions of Eritrea in 2015. This activated the Ministry of Health to conduct an initial exploratory investigation. The main objective of the investigation was to confirm the sensitivity of the RDTs in the field in microscopically confirmed malaria cases, identify the possible causes of the failure and recommend further actions to be taken.MethodsA team composed of the National Malaria Control Programme, National Medicines and Food Administration and Laboratory Unit of the Ministry of Health was established to confirm the sensitivity of the SD Bioline® RDTs. A ‘Malaria RDT quality monitoring form’ was prepared and distributed to 13 health facilities selected on availability of microscopy services, experienced laboratory personnel and malaria endemicity, to carry out preliminary investigation on the suspected RDT quality defect. In parallel, field visits to central and regional medical warehouses as well as selected health facilities were conducted to assess the storage conditions, handling and operator procedures. Furthermore, joint field assessment was conducted with the manufacturer, SD Bioline RDTs. During the time frame of 15 July 2015 to 19 January 2016, 65 microscopically confirmed patients were tested with Malaria RDTs SD Bioline Pf/Pv/Mixed Combo cassettes.ResultsA total of 65 blood specimens (50 P. falciparum, 13 Plasmodium vivax and 2 mixed) confirmed microscopically were tested against the available lots of malaria RDTs. Out of the 50 P. falciparum infected blood specimens, only 10 were confirmed positive with all the lots of PfHRP-2 detecting RDTs making the false negativity rate at 80% [41/51]. The false negative result for RDT targeting PfHRP2 antigen ranged from 65% [11/17] in Gash Barka region to 100% [12/12] in Northern Red Sea Region. In addition, supervisory visits undertaken by the study team have ruled out storage, handling and operator errors as causes of false negative results as the above parameter were found to be up to standards.ConclusionThe investigation confirmed high false-negativity rate in PfHRP-2 detecting RDTs and has ruled out quality of RDTs, storage, handling and operator error as causes of the reported problem. Therefore, molecular characterization of the P. falciparum is highly recommended to explore if parasite characteristics can be considered as causes of false negative results.

Pfhrp2-Deleted Plasmodium falciparum Parasites in the Democratic Republic of the Congo: A National Cross-sectional Survey.

Rapid diagnostic tests (RDTs) account for more than two-thirds of malaria diagnoses in Africa. Deletions of the Plasmodium falciparum hrp2 (pfhrp2) gene cause false-negative RDT results and have never been investigated on a national level. Spread of pfhrp2-deleted P. falciparum mutants, resistant to detection by HRP2-based RDTs, would represent a serious threat to malaria elimination efforts. Using a nationally representative cross-sectional study of 7,137 children under five years of age from the Democratic Republic of Congo (DRC), we tested 783 subjects with RDT-/PCR+ results using PCR assays to detect and confirm deletions of the pfhrp2 gene. Spatial and population genetic analyses were employed to examine the distribution and evolution of these parasites. We identified 149 pfhrp2-deleted parasites, representing 6.4% of all P. falciparum infections country-wide (95% confidence interval 5.1-8.0%). Bayesian spatial analyses identified statistically significant clustering of pfhrp2 deletions near Kinshasa and Kivu. Population genetic analysis revealed significant genetic differentiation between wild-type and pfhrp2-deleted parasite populations (GST = .046, p ≤ .00001). Pfhrp2-deleted P. falciparum is a common cause of RDT-/PCR+ malaria among asymptomatic children in the DRC and appears to be clustered within select communities. Surveillance for these deletions is needed, and alternatives to HRP2-specific RDTs may be necessary.

Multiple genetic origins of histidine-rich protein 2 gene deletion in Plasmodium falciparum parasites from Peru

The majority of malaria rapid diagnostic tests (RDTs) detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), encoded by the pfhrp2 gene. Recently, P. falciparum isolates from Peru were found to lack pfhrp2 leading to false-negative RDT results. We hypothesized that pfhrp2-deleted parasites in Peru derived from a single genetic event. We evaluated the parasite population structure and pfhrp2 haplotype of samples collected between 1998 and 2005 using seven neutral and seven chromosome 8 microsatellite markers, respectively. Five distinct pfhrp2 haplotypes, corresponding to five neutral microsatellite-based clonal lineages, were detected in 1998-2001; pfhrp2 deletions occurred within four haplotypes. In 2003-2005, outcrossing among the parasite lineages resulted in eight population clusters that inherited the five pfhrp2 haplotypes seen previously and a new haplotype; pfhrp2 deletions occurred within four of these haplotypes. These findings indicate that the genetic origin of pfhrp2 deletion in Peru was not a single event, but likely occurred multiple times.

False-Negative Rapid Diagnostic Tests for Malaria and Deletion of the Histidine-Rich Repeat Region of the hrp2 Gene †

We identified 480 persons with positive thick smears for asexual Plasmodium falciparum parasites, of whom 454 had positive rapid diagnostic tests (RDTs) for the histidine-rich protein 2 (HRP2) product of the hrp2 gene and 26 had negative tests. Polymerase chain reaction (PCR) amplification for the histidine-rich repeat region of that gene was negative in one-half (10/22) of false-negative specimens available, consistent with spontaneous deletion. False-negative RDTs were found only in persons with asymptomatic infections, and multiplicities of infection (MOIs) were lower in persons with false-negative RDTs (both P < 0.001). These results show that parasites that fail to produce HRP2 can cause patent bloodstream infections and false-negative RDT results. The importance of these observations is likely to increase as malaria control improves, because lower MOIs are associated with false-negative RDTs and false-negative RDTs are more frequent in persons with asymptomatic infections. These findings suggest that the use of HRP2-based RDTs should be reconsidered.