Comparison of prevalence estimates of pfhrp2 and pfhrp3 deletions in Plasmodium falciparum determined by conventional PCR and multiplex qPCR and implications for surveillance and monitoring.

OBJECTIVES: The accuracy of malaria rapid diagnostic tests is threatened by Plasmodium falciparum with pfhrp2/3 deletions. This study compares gene deletion prevalence determined by multiplex real time polymerase chain reaction (qPCR) and conventional polymerase chain reaction (cPCR) using existing samples with clonality previously determined by microsatellite genotyping. METHODS: Multiplex qPCR was used to estimate prevalence of pfhrp2/3 deletions in three sets of previously collected patient samples from Eritrea and Peru. The qPCR was validated by multiplex digital polymerase chain reaction. Sample classification was compared with cPCR, and receiver operating characteristic curve analysis was used to determine the optimal ΔCq threshold that aligned the results of the two assays. RESULTS: qPCR classified 75% (637 of 849) of samples as single, and 212 as mixed-pfhrp2/3 genotypes, with a positive association between clonality and proportion of mixed-pfhrp2/3 genotype samples. The sample classification agreement between cPCR and qPCR was 75.1% (95% confidence interval [CI] 68.6-80.7%) and 47.8% (95% CI 38.9-56.9%) for monoclonal and polyclonal infections. The qPCR prevalence estimates of pfhrp2/3 deletions showed almost perfect (κ = 0.804, 95% CI 0.714-0.895) and substantial agreement (κ = 0.717, 95% CI 0.562-0.872) with cPCR for Peru and 2016 Eritrean samples, respectively. For 2019 Eritrean samples, the prevalence of double pfhrp2/3 deletions was approximately two-fold higher using qPCR. The optimal threshold for matching the assay results was ΔCq = 3. CONCLUSIONS: Multiplex qPCR and cPCR produce comparable estimates of gene deletion prevalence when monoclonal infections dominate; however, qPCR provides higher estimates where multi-clonal infections are common.

Increasing Prevalence of Artemisinin-Resistant HRP2-Negative Malaria in Eritrea.

BACKGROUND: Although the clinical efficacy of antimalarial artemisinin-based combination therapies in Africa remains high, the recent emergence of partial resistance to artemisinin in Plasmodium falciparum on the continent is troubling, given the lack of alternative treatments. METHODS: In this study, we used data from drug-efficacy studies conducted between 2016 and 2019 that evaluated 3-day courses of artemisinin-based combination therapy (artesunate-amodiaquine or artemether-lumefantrine) for uncomplicated malaria in Eritrea to estimate the percentage of patients with day-3 positivity (i.e., persistent P. falciparum parasitemia 3 days after the initiation of therapy). We also assayed parasites for mutations in Pfkelch13 as predictive markers of partial resistance to artemisinin and screened for deletions in hrp2 and hrp3 that result in variable performance of histidine rich protein 2 (HRP2)-based rapid diagnostic tests for malaria. RESULTS: We noted an increase in the percentage of patients with day-3 positivity from 0.4% (1 of 273) in 2016 to 1.9% (4 of 209) in 2017 and 4.2% (15 of 359) in 2019. An increase was also noted in the prevalence of the Pfkelch13 R622I mutation, which was detected in 109 of 818 isolates before treatment, from 8.6% (24 of 278) in 2016 to 21.0% (69 of 329) in 2019. The odds of day-3 positivity increased by a factor of 6.2 (95% confidence interval, 2.5 to 15.5) among the patients with Pfkelch13 622I variant parasites. Partial resistance to artemisinin, as defined by the World Health Organization, was observed in Eritrea. More than 5% of the patients younger than 15 years of age with day-3 positivity also had parasites that carried Pfkelch13 R622I. In vitro, the R622I mutation conferred a low level of resistance to artemisinin when edited into NF54 and Dd2 parasite lines. Deletions in both hrp2 and hrp3 were identified in 16.9% of the parasites that carried the Pfkelch13 R622I mutation, which made them potentially undetectable by HRP2-based rapid diagnostic tests. CONCLUSIONS: The emergence and spread of P. falciparum lineages with both Pfkelch13-mediated partial resistance to artemisinin and deletions in hrp2 and hrp3 in Eritrea threaten to compromise regional malaria control and elimination campaigns. (Funded by the Bill and Melinda Gates Foundation and others; Australian New Zealand Clinical Trials Registry numbers, ACTRN12618001223224, ACTRN12618000353291, and ACTRN12619000859189.).

Epidemiology of mutant Plasmodium falciparum parasites lacking histidine-rich protein 2/3 genes in Eritrea 2 years after switching from HRP2-based RDTs.

Eritrea was the first African country to complete a nationwide switch in 2016 away from HRP2-based RDTs due to high rates of false-negative RDT results caused by Plasmodium falciparum parasites lacking hrp2/hrp3 genes. A cross-sectional survey was conducted during 2019 enrolling symptomatic malaria patients from nine health facilities across three zones consecutively to investigate the epidemiology of P. falciparum lacking hrp2/3 after the RDT switch. Molecular analyses of 715 samples revealed the overall prevalence of hrp2-, hrp3-, and dual hrp2/3-deleted parasites as 9.4% (95%CI 7.4-11.7%), 41.7% (95% CI 38.1-45.3%) and 7.6% (95% CI 5.8-9.7%), respectively. The prevalence of hrp2- and hrp3-deletion is heterogeneous within and between zones: highest in Anseba (27.1% and 57.9%), followed by Gash Barka (6.4% and 37.9%) and Debub zone (5.2% and 43.8%). hrp2/3-deleted parasites have multiple diverse haplotypes, with many shared or connected among parasites of different hrp2/3 status, indicating mutant parasites have likely evolved from multiple and local parasite genetic backgrounds. The findings show although prevalence of hrp2/3-deleted parasites is lower 2 years after RDT switching, HRP2-based RDTs remain unsuitable for malaria diagnosis in Eritrea. Continued surveillance of hrp2/3-deleted parasites in Eritrea and neighbouring countries is required to monitor the trend.

Novel sampling methods for monitoring Anopheles arabiensis from Eritrea.

BACKGROUND: Studies comparing novel collection methods for host seeking and resting mosquitoes A. arabiensis were undertaken in a village in Eritrea. Techniques included an odor baited trap, a novel tent-trap, human landing collection and three methods of resting collection. A technique for the collection of mosquitoes exiting vegetation is also described. Pre-gravid rates were determined by dissection of host seeking insects and post-prandial egg development among insects collected resting. RESULTS: Overall 5,382 host-seeking, 2,296 resting and 357 A. arabiensis exiting vegetation were collected. The Furvela tent-trap was the most efficient, risk-free method for the collection of outdoor host-seeking insects, whilst the Suna trap was the least effective method. Mechanical aspirators (the CDC backpack or the Prokopack aspirator) were superior to manual aspiration in a dark shelter but there was no advantage over manual aspiration in a well-lit one. An estimated two-thirds of newly-emerged mosquitoes went through a pre-gravid phase, feeding twice before producing eggs. Mosquitoes completed gonotrophic development in a dark shelter but left a well-lit shelter soon after feeding. One blood-fed female marked in the village was recaptured 2 days after release exiting vegetation close to the oviposition site and another, shortly after oviposition, attempting to feed on a human host 3 days after release. Exit rates of males from vegetation peaked 3 min after the initial male had left. Unfed and gravid females exited approximately 6 min after the first males. CONCLUSIONS: Furvela tent-traps are suitable for the collection of outdoor biting A. arabiensis in Eritrea whilst the Prokopack sampler is the method of choice for the collection of resting insects. Constructing well-lit, rather than dark, animal shelters, may encourage otherwise endophilic mosquitoes to leave and so reduce their survival and hence their vectorial capacity.

Prevalence of CYP2C8*2 and *3 among Eritreans and its Potential Impact on Artesunate/Amodiaquine Treatment.

BACKGROUND: In Eritrea, artesunate-amodiaquine is the first-line treatment against uncomplicated malaria. Amodiaquine, which is mainly bio-transformed by CYP2C8, is known to be associated with adverse events of different severity. Extrapyramidal events are among the less common but have been reported with non-negligible frequency in Eritrea. This study was conducted to investigate the allele frequencies of CYP2C8*2 and *3, both associated with decreased amodiaquine metabolism, among the Eritrean population. METHODS: During September-November 2018, dried blood samples from 380 participants and 17 patients who previously had experienced extrapyramidal symptoms following treatment of artesunate-amodiaquine were collected and PCR-RFLP genotyped for CYP2C8*2 and *3. RESULTS: The allele frequencies of CYP2C8*2 and *3 were determined as 5.9% (95% CI: 4.4-7.8) and 4.6% (95% CI: 3.2-6.3), respectively. Four out of the 17 patients with extrapyramidal reactions showed to be carriers of the alleles. CONCLUSION: CYP2C8*2 and *3 frequencies among Eritreans were found to be intermediate between the documented for Caucasian and African populations. These findings, along with the alleles not being decisive for the occurrence of extrapyramidal events, might be of importance regarding the amodiaquine-containing malaria treatment in Eritrea. Furthermore, it suggests a significant proportion of slow amodiaquine metabolizers in the Sahel region, information of potential interest in the context of amodiaquine-involving seasonal malaria chemoprevention.

A novel multiplex qPCR assay for detection of Plasmodium falciparum with histidine-rich protein 2 and 3 (pfhrp2 and pfhrp3) deletions in polyclonal infections.

BACKGROUND: Many health facilities in malaria endemic countries are dependent on Rapid diagnostic tests (RDTs) for diagnosis and some National Health Service (NHS) hospitals without expert microscopists rely on them for diagnosis out of hours. The emergence of P. falciparum lacking the gene encoding histidine-rich protein 2 and 3 (HRP2 and HRP3) and escaping RDT detection threatens progress in malaria control and elimination. Currently, confirmation of RDT negative due to the deletion of pfhrp2 and pfhrp3, which encodes a cross-reactive protein isoform, requires a series of PCR assays. These tests have different limits of detection and many laboratories have reported difficulty in confirming the absence of the deletions with certainty. METHODS: We developed and validated a novel and rapid multiplex real time quantitative (qPCR) assay to detect pfhrp2, pfhrp3, confirmatory parasite and human reference genes simultaneously. We also applied the assay to detect pfhrp2 and pfhrp3 deletion in 462 field samples from different endemic countries and UK travellers. RESULTS: The qPCR assay demonstrated diagnostic sensitivity of 100% (n = 19, 95% CI= (82.3%; 100%)) and diagnostic specificity of 100% (n = 31; 95% CI= (88.8%; 100%)) in detecting pfhrp2 and pfhrp3 deletions. In addition, the assay estimates P. falciparum parasite density and accurately detects pfhrp2 and pfhrp3 deletions masked in polyclonal infections. We report pfhrp2 and pfhrp3 deletions in parasite isolates from Kenya, Tanzania and in UK travellers. INTERPRETATION: The new qPCR is easily scalable to routine surveillance studies in countries where P. falciparum parasites lacking pfhrp2 and pfhrp3 are a threat to malaria control.

Retrospective data analyses of social and environmental determinants of malaria control for elimination prospects in Eritrea.

BACKGROUND: The present study focuses on both long- and short-term malaria transmission in Eritrea and investigates the risk factors. Annual aggregates of information on malaria cases, deaths, diagnostics and control interventions from 2001 to 2008 and monthly reported data from 2009 to 2017 were obtained from the National Malaria Control Programme. We used a generalized linear regression model to examine the associations among total malaria cases, death, insecticide-treated net coverage, indoor residual spraying and climatic parameters. RESULTS: Reduction in malaria mortality is demonstrated by the milestone margins of over 97% by the end of 2017. Malaria incidence likewise declined during the period (from 33 to 5 per 1000 population), representing a reduction of about 86% (R2 = 0.3) slightly less than the decline in mortality. The distribution of insecticide treated nets generally declined between 2001 and 2014 (R2 = 0.16) before increasing from 2015 to 2017, while the number of people protected by indoor residual spraying slightly increased (R2 = 0.27). Higher rainfall was significantly associated with an increased number of malaria cases. The covariates rainfall and temperature are a better pair than IRS and LLIN to predict incidences. On the other hand, IRS and LLIN is a more significant pair to predict mortality cases. CONCLUSIONS: While Eritrea has made significant progress towards malaria elimination, this progress should be maintained and further improved. Distribution, coverage and utilization of malaria control and elimination tools should be optimized and sustained to safeguard the gains made. Additionally, consistent annual performance evaluation of malaria indicators would ensure a continuous learning process from gains/threats of epidemics and resurgence in regions already earmarked for elimination.

Mosquito feeding behavior and how it influences residual malaria transmission across Africa.

The antimalarial efficacy of the most important vector control interventions-long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS)-primarily protect against mosquitoes' biting people when they are in bed and indoors. Mosquito bites taken outside of these times contribute to residual transmission which determines the maximum effectiveness of current malaria prevention. The likelihood mosquitoes feed outside the time of day when LLINs and IRS can protect people is poorly understood, and the proportion of bites received outdoors may be higher after prolonged vector control. A systematic review of mosquito and human behavior is used to quantify and estimate the public health impact of outdoor biting across Africa. On average 79% of bites by the major malaria vectors occur during the time when people are in bed. This estimate is substantially lower than previous predictions, with results suggesting a nearly 10% lower proportion of bites taken at the time when people are beneath LLINs since the year 2000. Across Africa, this higher outdoor transmission is predicted to result in an estimated 10.6 million additional malaria cases annually if universal LLIN and IRS coverage was achieved. Higher outdoor biting diminishes the cases of malaria averted by vector control. This reduction in LLIN effectiveness appears to be exacerbated in areas where mosquito populations are resistant to insecticides used in bed nets, but no association was found between physiological resistance and outdoor biting. Substantial spatial heterogeneity in mosquito biting behavior between communities could contribute to differences in effectiveness of malaria control across Africa.

'We like it wet': a comparison between dissection techniques for the assessment of parity in Anopheles arabiensis and determination of sac stage in mosquitoes alive or dead on collection.

BACKGROUND: The determination of parous rates in mosquitoes, despite numerous shortcomings, remains a tool to evaluate the effectiveness of control programs and to determine vectorial capacity in malaria vectors. Two dissection techniques are used for this. For one, the tracheoles of dried ovaries are examined with a compound microscope and in the other the follicular stalk of ovaries is examined, wet, with a stereomicroscope. The second method also enables the sac stage of parous insects (which provides information on the duration of the oviposition cycle) and the mated status of insects to be determined. Despite widespread use the two techniques have not previously been compared. METHODS: We compared the two dissection techniques using Anopheles arabiensis, collected with a tent-trap in Eritrea. The paired ovaries were removed in water and one was examined by each method. From a separate set of dissections from Tanzania, we also determined if the sac stages of Anopheles gambiae s.l. (83% of 183 identified by PCR being Anopheles arabiensis the remainder being A. gambiae) that were alive on collection were different to those that died on collection and what the implications for vectorial capacity estimation might be. RESULTS: Seven per cent of the dry ovaries could not be classified due to granulation (yolk) in the ovariole that obscured the tracheoles. The sensitivity of the dry dissection was 88.51% (CI [79.88-94.35%]) and the specificity was 93.55% (CI [87.68-97.17%]) among the 211 ovaries that could be classified by the dry technique and compared to the ovaries dissected wet. 1,823 live and 1,416 dead from Furvela tent-traps, CDC light-trap and window-trap collections were dissected 'wet' from Tanzania. In these collections parous insects were more likely to die compared to nulliparous ones. The proportion of parous mosquitoes with 'a' sacs (indicative of recent oviposition) was significantly greater in insects that were dead (0.36) on collection in the morning compared to those that were alive (0.12) (Chi square 138.93, p < 0.001). There was a preponderance of newly emerged virgin insects in the outdoor collection (Chi sq = 8.84, p = 0.003). CONCLUSIONS: In anophelines the examination of mosquito ovaries using transmitted light in a 'wet' dissection is a more useful and informative technique than examination of dry ovaries. In order to correctly estimate the duration of the oviposition cycle mosquitoes should be dissected as soon as possible after collection. Younger insects were more likely to attempt to feed outdoors rather than indoors.

Major Threat to Malaria Control Programs by Plasmodium falciparum Lacking Histidine-Rich Protein 2, Eritrea.

False-negative results for Plasmodium falciparum histidine-rich protein (HRP) 2-based rapid diagnostic tests (RDTs) are increasing in Eritrea. We investigated HRP gene 2/3 (pfhrp2/pfhrp3) status in 50 infected patients at 2 hospitals. We showed that 80.8% (21/26) of patients at Ghindae Hospital and 41.7% (10/24) at Massawa Hospital were infected with pfhrp2-negative parasites and 92.3% (24/26) of patients at Ghindae Hospital and 70.8% (17/24) at Massawa Hospital were infected with pfhrp3-negative parasites. Parasite densities between pfhrp2-positive and pfhrp2-negative patients were comparable. All pfhrp2-negative samples had no detectable HRP2/3 antigen and showed negative results for HRP2-based RDTs. pfhrp2-negative parasites were genetically less diverse and formed 2 clusters with no close relationships to parasites from Peru. These parasites probably emerged independently by selection in Eritrea. High prevalence of pfhrp2-negative parasites caused a high rate of false-negative results for RDTs. Determining prevalence of pfhrp2-negative parasites is urgently needed in neighboring countries to assist case management policies.

Artesunate/Amodiaquine-Induced Acute Extrapyramidal Reactions in Children and Younger Adults: Case Series Assessment.

INTRODUCTION: Several studies conducted in African countries reported the artesunate and amodiaquine (AS/AQ) tablet as a safe and well-tolerated anti-malarial drug in children and younger adults. The aim of this case series assessment was to assess the causal relationship between the AS/AQ tablet and extrapyramidal reactions in children and younger adults and to investigate the factor(s) predisposing to the adverse drug reactions. METHODS: The causal relationship of all the cases was first assessed individually using the Naranjo Probability Scale and then subjected to a case series assessment using Austin Bradford-Hill criteria. RESULTS: A total of 43 acute extrapyramidal reactions associated with the AS/AQ tablet were reported between 2012 and 16 November, 2015 to the Eritrean Pharmacovigilance Centre. The causality was found to be probable or highly probable for 33 (76.7 %) of the cases and the rest (10; 23.3 %) of the cases had a possible causal association. The extrapyramidal reactions had more or less similar clinical features in most of the cases and were characterized by abnormal involuntary contractions of muscles. The median age and body weight of the cases were 15 years and 40 kg, respectively, and 70 % of them were males. 90.7% of the reactions manifested in children and younger adults (aged <26 years). In most of the cases, reactions manifested in the third day from the start of treatment and 88.3 % of cases were hospitalized. CONCLUSION: The causal relationship between the AS/AQ tablet and extrapyramidal reactions in children and younger adults was found to be apparent and possibly owing to dose accumulation or an overdose of amodiaquine.

Consolidating strategic planning and operational frameworks for integrated vector management in Eritrea.

BACKGROUND: Contemporary malaria vector control relies on the use of insecticide-based, indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs). However, malaria-endemic countries, including Eritrea, have struggled to effectively deploy these tools due technical and operational challenges, including the selection of insecticide resistance in malaria vectors. This manuscript outlines the processes undertaken in consolidating strategic planning and operational frameworks for vector control to expedite malaria elimination in Eritrea. CASE DESCRIPTION: The effort to strengthen strategic frameworks for vector control in Eritrea was the 'case' for this study. The integrated vector management (IVM) strategy was developed in 2010 but was not well executed, resulting in a rise in malaria transmission, prompting a process to redefine and relaunch the IVM strategy with integration of other vector borne diseases (VBDs) as the focus. The information sources for this study included all available data and accessible archived documentary records on malaria vector control in Eritrea. Structured literature searches of published, peer-reviewed sources using online, scientific, bibliographic databases, Google Scholar, PubMed and WHO, and a combination of search terms were utilized to gather data. The literature was reviewed and adapted to the local context and translated into the consolidated strategic framework. DISCUSSION: In Eritrea, communities are grappling with the challenge of VBDs posing public health concerns, including malaria. The global fund financed the scale-up of IRS and LLIN programmes in 2014. Eritrea is transitioning towards malaria elimination and strategic frameworks for vector control have been consolidated by: developing an integrated vector management (IVM) strategy (2015-2019); updating IRS and larval source management (LSM) guidelines; developing training manuals for IRS and LSM; training of national staff in malaria entomology and vector control, including insecticide resistance monitoring techniques; initiating the global plan for insecticide resistance management; conducting needs' assessments and developing standard operating procedure for insectaries; developing a guidance document on malaria vector control based on eco-epidemiological strata, a vector surveillance plan and harmonized mapping, data collection and reporting tools. CONCLUSION: Eritrea has successfully consolidated strategic frameworks for vector control. Rational decision-making remains critical to ensure that the interventions are effective and their choice is evidence-based, and to optimize the use of resources for vector control. Implementation of effective IVM requires proper collaboration and coordination, consistent technical and financial capacity and support to offer greater benefits.

Gains attained in malaria control coverage within settings earmarked for pre-elimination: malaria indicator and prevalence surveys 2012, Eritrea.

BACKGROUND: Eritrea, like most countries in sub-Saharan Africa, has expended much effort towards malaria control with the view of transitioning from reduction of the disease burden to elimination. This paper reports on the level of achievement as highlighted by the follow-on, malaria-endemic area representative, survey that aimed to provide data and to assess progress on malaria indicators and parasite prevalence at household level across the country. METHODS: In 2012, data were collected using a two-stage stratified cluster random sample of 1887 households in 96 clusters (villages in rural areas and census enumeration areas in urban centers) during a malaria indicator and prevalence survey in Eritrea. The survey determined parasite prevalence in vulnerable population groups and evaluated coverage, use and access to malaria control services. Standardized Roll-Back Malaria Monitoring and Evaluation Reference Group household and women's questionnaires were adapted to the local situation and used for collection of data that were analysed and summarized using descriptive statistics. RESULTS: The results of the survey showed that 90% (95% CI 89-91) of households owned at least one mosquito net. The proportion of the population with access to an insecticide-treated net (ITN) in their household was 55% (95% CI 54-56). The utilization of ITNs was 67% (95% CI 65-70) for children under 5 years and 60% (95% CI 58-63) for pregnant women (OR: 0. 73(95% CI 0.62-0.85); P = 0.52). Only 28% (95% CI 26-30) of households were covered by indoor residual spraying (IRS) the previous year with significant heterogeneity by zoba (Debub 50 % (95% CI 45-54) vs Gash Barka 32 % (95% CI 28-36); OR = 0. 47 (95% CI 0.36-0.61), P = 0.05). Malaria parasite prevalence was low; 1.1% (95% CI 0.9-1.3) in the general population and 1.4% (95% CI 1.0-2.0) in children under five and 0.7% (95% CI 0.4-1.1) among women aged 15-49 years. Only 19% (95% CI 15-26) of children under five had fever in the 2 weeks preceding the survey, with 61% (95% CI 54.1-67.1) seeking treatment from a health facility. Data on knowledge levels show that 92% reported that malaria is transmitted by mosquitoes, 92% mentioned that the use of mosquito nets could prevent malaria, 47% knew malaria prevention medication, 83% cited fever as a sign and symptom of malaria, and 35% had heard or seen malaria awareness messages. CONCLUSION: Notwithstanding confounders, the observed low malaria parasite prevalence could be associated with malaria intervention coverage, access and utilization as well as high and equitable knowledge levels in the population. This indicates that Eritrea is on the right track towards pre-elimination. However, technical and infrastructure capacity should be strengthened to facilitate implementation, surveillance, monitoring, and evaluation.

Evaluating indoor residual spray for reducing malaria infection prevalence in Eritrea: results from a community randomized control trial.

This paper examines the relationship between indoor residual spray (IRS) and malaria parasite infection in Gash Barka Zone, Eritrea, an area with near universal coverage of insecticide treated bednets (ITN) and already low malaria parasite prevalence. A community randomized control trial was conducted in 2009. Malaria parasite infection prevalence was 0.5% [95% confidence interval (CI): 0.37-0.78%], with no significant difference detected between treatment and control areas. ITN possession remains high, with over 70% of households reporting ITN ownership [95% CI: 68.4-72.9]. ITN use among individuals within ITN-owning households was just under half [46.7% (95% CI: 45.4-48.0)]. Slight differences in ITN possession and use were detected between treatment and control areas. There was no significant difference in malaria parasite infection prevalence among individuals in households with ≥1 ITN compared to those in households without ITNs, nor among individuals reporting ITN use. Among individuals in ITN-owning households, sleeping under an ITN offered no statistically significant protection from malaria parasite infection. Community participation in environmental and larval habitat management activities was low: 17.9% (95% CI: 16.0-19.7). It is likely that IRS, larval habitat management and ITN distribution alone may be insufficient to interrupt transmission without corresponding high ITN use, sustained IRS application in areas where infections are clustered, and promptly seeking laboratory diagnosis and treatment of all fevers. Eritrea is ready for elimination, irrespective of inconclusive impact evaluation results.