Plasmodium falciparum apical membrane antigen 1 (AMA1) have failed due to extensive polymorphism in AMA1. To assess the strain-specificity of antibody responses to malaria infection and AMA1 vaccination, we designed protein and peptide microarrays representing hundreds of unique AMA1 variants. Following clinical malaria episodes, children had short-lived, sequence-independent increases in average whole-protein seroreactivity, as well as strain-specific responses to peptides representing diverse epitopes. Vaccination resulted in dramatically increased seroreactivity to all 263 AMA1 whole-protein variants. High-density peptide analysis revealed that vaccinated children had increases in seroreactivity to four distinct epitopes that exceeded responses to natural infection. A single amino acid change was critical to seroreactivity to peptides in a region of AMA1 associated with strain-specific vaccine efficacy. Antibody measurements using whole antigens may be biased towards conserved, immunodominant epitopes. Peptide microarrays may help to identify immunogenic epitopes, define correlates of vaccine protection, and measure strain-specific vaccine-induced antibodies.Vaccines based on  Plasmodium falciparum apical membrane antigen 1 (AMA1) is a malaria parasite surface protein involved in red blood cell invasion1. AMA1 has been a leading target for vaccine development because of its high antigenicity and the ability of AMA1 antibodies to inhibit parasite growth both in vitro and in non-human primates6. However, in vitro and molecular epidemiological studies provided early evidence that AMA1 exhibits immune evasion that is both domain- and sequence-specific12. The AMA1 protein contains a hydrophobic cleft that is the binding site of red blood cell invasion machinery16. Synthetic peptides that bind to this hydrophobic cleft prevent interaction with the red blood cell invasion complex, obstructing parasite invasion18. The hydrophobic cleft is in the first of AMA1’s three domains, surrounded by six flexible, polymorphic loops thought to prevent host antibodies from disrupting the formation of the invasion complex19. Moreover, polymorphisms in a region known as the cluster 1 loop of domain 1 have been shown to affect the binding of inhibitory monoclonal antibodies in vitro20. A monovalent AMA1 subunit vaccine tested in a phase 2 clinical trial in Malian children did not provide significant protection against all clinical malaria21. However, the vaccine demonstrated significant efficacy against clinical malaria caused by strains with the vaccine-type (3D7 strain) ama1 sequence at the cluster 1 loop22.ama1 consists of single nucleotide polymorphisms at more than 60 loci encoding amino acid changes throughout all three domains of the ectodomain, but concentrated in domain 19. We previously identified 214 unique ama1 ectodomain sequences among 506 single or predominant clone P. falciparum infections9 in Bandiagara, the town in central Mali where the AMA1 vaccine was tested in adults23 and children24. A prototype diversity-reflecting protein microarray that included 58 AMA1 variants showed increases in both lifetime and seasonal magnitude and breadth of anti-AMA1 antibodies in malaria-exposed Malian children and adults living in Bandiagara, where malaria transmission is intense but highly seasonal25. Here we report the use of an expanded protein microarray with 263 unique AMA1 variants isolated from the AMA1 vaccine trial site, as well as a high-density peptide array populated with overlapping 16-mer AMA1 peptides derived from our field isolates and publicly available sequences. Our aim was to examine the effects of both natural P. falciparum parasite exposure and AMA1 vaccination on the breadth and magnitude of strain-specific and epitope-specific antibody responses to AMA1.Diversity in 6 randomly-selected children were probed on a separate, mutation scan peptide microarray consisting of the hypervariable cluster 1 loop region.Sera were randomly selected from participants in a 3D7-based AMA1 vaccine trial conducted in Bandiagara, Mali to be probed on the whole protein microarray. Forty (40), AMA1-vaccinated children and 20 AMA1-vaccinated Malian adults with sera corresponding to pre-vaccination and 90 days post first AMA1 vaccination were selected. Additionally, 40 control-vaccinated Malian children and 20 control-vaccinated Malian adults were randomly selected, with sera corresponding pre-, peak, and post malaria season and probed on the whole-protein array. Sera from 11 North-American malaria-naïve blood donors were also probed on the whole protein array as malaria exposure negative controls. Sera from a subset of these participants were probed on the high-density peptide array, including 10 AMA1-vaccinated Malian children, 10 control-vaccinated Malian children, 10 control-vaccinated Malian adults, and five North American controls at the same vaccine and season-relative time points. Of the group of 10 AMA1-vaccinated Malian childrens’ samples run on the high-density peptide microarray, pre-and post AMA1-vaccination sera from sixp < 0.001, Mann-Whitney test, Fig. p < 0.001, Mann Whitney test, Supplementary Fig. p < 0.05, p <  0.02, respectively, Mann Whitney test, Fig. At the beginning of the malaria transmission season, the mean seroreactivity to the 263 AMA1 variants on the protein array was 81% higher in unvaccinated adults than unvaccinated children, and responses in both cohorts were greater than in North American malaria-naïve controls (26 were as follows: Domain 1 (positions #83–303), the largest of the three domains, was represented by 4,150 unique 16mer peptides derived from available sequence data and was more polymorphic than domains 2 (positions #304–418) and 3 (positions #419–546). Domains 2 and 3 were represented by 1,507 and 1,707 peptides, respectively. Both immunoreactive epitopes and non-reactive regions were found across the full length of AMA1 .Sera from unvaccinated Malian adults had greater breadth of serorecognition compared to unvaccinated Malian children to all three AMA1 ectodomains and the intracellular cytosolic region individually . Eight children did not have a positive malaria blood sample during follow up, and had protein microarray data for all three time points. Sera from infected control-vaccinated Malian children showed significant increases in median magnitude of mean seroreactivity to 263 AMA1 proteins from pre- to peak-season, and pre- to post-season, but no difference from peak- to post-season , compared to an 11% increase in children in the control group during the same time period . MAb 479 A is described as a <30% inhibitory antibody of malaria parasite FVO, and mAb 480 A has no known inhibitory activity. The exact binding locations of mAb 479 A and 480 A are not known. The binding loci for N3 negative control mAb DEN3; raised against dengue virus serotype 3, did not bind to AMA1 variants above empty-vector control features, yet bound to pan-IgG positive control features printed on the array  and one control Dengue virus monoclonal antibody to demonstrate strain-specificity on the whole-protein microarray platform. Monoclonal antibody 5A6, a known strain-specific, malaria inhibitory antibody that binds to a strain-specific domain-2 epitope, bound significantly to five AMA1 variants on the array above Dengue virus mAb control. The magnitude of seroreactivity to AMA1 variants of mAb MRA-479A (N3-2D9) and MRA-480A (N3-1D7) were highly correlated  sera for this subset of unvaccinated Malian children, the magnitude of seroreactivity to peptides matching the infecting strain(s) was compared to the seroreactivity to the peptide at the same locus that matched the reference strain 3D7 and reported as a ratio Fig. . OverallP. falciparum parasites expressing diverse AMA1 variants. To examine the variability in seroreactivity to epitopes with differing amino acid content we plotted the minimum, median, and maximum mean seroreactivity to variant peptides located at the same amino acid positions across the length of the AMA1 sequence, for ten control-vaccinated Malian adult sera probed on the high-density peptide array  in position 197 (E197) with a mean normalized signal of 4.75 . This subset of children were randomly selected from the subset of ten children whose sera were run on the peptide array. Six children were chosen due to the mutation scan array design, which allowed 12 samples (six pre- and six post-vaccination) to be probed on one slide, and no statistical power assessment was performed .75 Fig. . SubstitThis study used diversity-reflecting protein and peptide microarrays to understand naturally-acquired and vaccine-induced antibody responses to a highly polymorphic malaria surface protein and leading vaccine antigen. Natural infection gave rise to both short-lived, broadly cross-reactive antibodies as well as strain-specific antibody responses targeting diverse epitopes previously associated with both natural and vaccine-induced clinical immunity. Contrary to expectation, vaccination with a monovalent blood-stage vaccine that elicited strain-specific efficacy was followed by extremely broad and strong recognition of hundreds of variants of the vaccine antigen, as well as increases in seroreactivity to four distinct epitopes.E. coli-based transcription/translation system, but this system has been validated as a means of measuring clinically relevant antibody responses29.Probing sera on the whole-protein microarray measured the binding of antibodies to tertiary structures with both conformational and discontinuous epitopes. A limitation of this platform is its inability to ensure proper folding of proteins expressed using a cell-free The observation that the whole protein variant 193.1, which was 100% identical to vaccine strain 3D7 at 62 polymorphic loci, was not the most seroreactive variant among AMA1-vaccinated children or adults, was surprising. This could be explained by imprecise folding of the protein as printed on the array, or variability in the concentration of antigen deposited on the array. All variants used in the printing of the arrays were obtained from the same cloning, expression and purification experiment, to rule-out within-protein batch effects. Antibody seroreactivity as measured on the whole-protein array represents the summation of the binding of a complex collection of polyclonal antibodies to both conserved and strain-specific epitopes, and is affected by recent and cumulative exposures to malaria parasites. It is possible that previous exposure to malarial parasite AMA1 could influence the strain-specific serological response to AMA1 vaccination. The protein array is unable to detect responses to individual regions of the molecule that are targeted preferentially in response to natural infection or AMA1 vaccination.Seroprofiles generated by probing sera on the high-density linear peptide array provide measurements of the antibody responses to individual epitopes, and the resolution to investigate the effect of single amino acid substitutions on antibody seroreactivity to these polymorphic epitopes. Using this platform, we measured the relative immunogenicity of domains and individual epitopes to uncover immunodominant regions. The flexible linear epitopes on the peptide array undoubtedly fail to represent some important conformational and discontinuous epitopes. With short linear peptides, it is also possible that short amino acid sequences are shared between different organisms, and not specific to malaria. However, combining the two methodologies provided a broader picture of antibody responses in population-based studies, with each platform’s strengths compensating to some degree for the limitations of the other.31. Infection by different malaria strains may lead to the generation of diverse antibodies that prevent new clinical malaria episodes upon re-exposure to previously encountered variant parasite antigens. This model is supported by molecular epidemiological evidence showing that children experiencing the second of two consecutive infections with genotyped AMA1 variants had a greater likelihood of clinical symptoms when that second infection differed from the preceding one at key polymorphic loci 9. Supporting this model of clinical immunity to malaria, in the present study, Malian adults with lifelong exposure had higher magnitude and breadth of serorecognition to AMA1 variants than Malian children.Clinical immunity to malaria is thought to be acquired only after an individual has been exposed to a variety of genetically distinct parasitesSurprisingly, the distribution of serorecognition in children was bimodal; children either recognized the majority of AMA1 whole-protein variants or just few. This phenomenon may be explained by high seroreactivity to the immunodominant cytosolic region of the AMA1 protein, which is more conserved. With each exposure to malaria parasites, seroreactivity to epitopes including those in the conserved cytosolic region may increase, eventually reaching a point at which children cross a threshold of seroreactivity to recognize the majority AMA1 whole-protein variants above background.Comparing seroprofiles of adults to those of children using both platforms permitted inference of which targets of AMA1 antibodies are correlated with protection against clinical malaria, with the important caveat that it is difficult to distinguish markers of protection from markers of exposure. Adults had higher magnitude seroreactivity to structural loops surrounding the binding site of the AMA1-RON2 invasion complex. Adults also recognized more diverse variants of these loops than did children, and were able to mount effective multi-strain antibody responses to a larger collection of diverse malaria parasites. This observation could lead to a better serological correlate of protection from clinical malaria disease that is not subject to spurious results due to the strain-specificity of antibodies to malaria structural epitopes.35. Our data show that diverse strain-specific antibody responses similarly wane quickly. We observed an increase in childrens’ antibody titers over the malaria season that peaked and then declined in the post-season as transmission ended. While the seasonal change in seroreactivity to AMA1 proteins was not significant for children overall, the subset of children who experienced a clinical malaria episode had a significant increase in AMA1 seroreactivity, with peak seroreactivity corresponding to the peak of the malaria transmission season.The initial sharp increase in AMA1 antibody titers as measured by ELISA using a reference antigen has been reported to wane within six weeks after a single malaria infection7. A sharp increase in seroreactivity was observed in recent infections within the immunodominant cytosolic region of AMA1 that waned with time. Studying the dynamics of seroreactivity to specific peptides could potentially yield sensitive serological biomarkers for malaria exposure within discrete time frames in the late stages of malaria elimination.Children had increased seroreactivity to all AMA1 whole-protein variants on the array after a clinical malaria episode, regardless of the strain of the infecting parasites. This may be explained by the large increase in seroreactivity to epitopes in the more conserved intracellular cytosolic component of the AMA1 molecule seen on the peptide array following clinical episodes. Antibodies targeting the 1e-loop in the hydrophobic cleft have demonstrated inhibitory activityA vaccine contains the ectodomain of only the laboratory reference strain 3D7 AMA1, and we therefore expected the magnitude of seroreactivity to the vaccine strain 3D7 AMA1 variant to be significantly higher than seroreactivity to other whole-protein AMA1 variants after vaccination. However, both children and adults had a broad increase in AMA1 seroreactivity on the whole-protein microarray following vaccination. Clinical protection was observed only when individuals were infected with a parasites homologous to the vaccine strain with respect to the protein sequence of the cluster 1 loop21. While there was significant variability in the magnitude of seroreactivity to different variants of AMA1 proteins, common protein motifs that correlated with vaccination or protection could not be identified. This may be due to the complexity of the polyclonal antibody response as measured on the whole protein array, with hundreds of competing antibodies binding to both variable and conserved regions of the molecule.The FMP2.1/AS02Peptide array analysis of sera from AMA1-vaccinated Malian children revealed the presence of at least four immunodominant epitopes, each with variable levels of amino acid conservation. The sum of seroreactivity to peptides within these four epitopes alone far eclipsed the seroreactivity to every other AMA1 peptide combined. This would explain the broad reactivity observed on the protein array: seroreactivity to any one of these four epitopes would obfuscate any sequence-dependent differences in antibody reactivity that may exist. Without these insights from the peptide array, probing sera from AMA1-vaccinated children on the whole-protein array alone would lead to the false conclusion that the vaccine had strain-transcending immunogenicity.P. falciparum infection differed from those measured after AMA1 vaccination in Malian children in the epitopes they preferentially targeted, likely in part because the vaccine was comprised of the AMA1 ectodomain without including the cytosolic region.While a protective vaccine does not necessarily need to generate antibodies mimicking those elicited by natural infection, it may be important to note the differences between seroprofiles in clinically immune individuals and those seen following vaccination. Our results showed that antibodies circulating in clinically immune Malian adults after a lifetime of 37.Despite the clear evidence from previous studies of strain-specific natural and vaccine-induced immunity to AMA1, strain-specificity of the antibodies generated after malaria infection and AMA1 vaccination could not be demonstrated using the whole-protein array. As noted above, this inability may be explained, in part, by the immunodominance and relative conservation of the cytosolic region. Antibodies targeting epitopes in the cytosolic region may overwhelm any detectable seroreactivity to epitopes displaying antigenic variation in domains 1–3. Conversely, probing monoclonal antibodies on the whole-protein microarray demonstrated the platform’s ability to analyze the breadth of cross-reactive inhibitory antibodies, which may facilitate discovery of protective epitopes and inform vaccine development. Notably, the strain-specificity of antibody responses to individual peptide variants was observed at the same loci among different individuals. These commonly recognized loci targeted by strain-specific antibodies included polymorphic structural loops previously implicated in natural and vaccine-induced allele-specific immunity as well as inhibitory monoclonal antibodiesAmong clinically immune Malian adults, there remained significant “lacunae” in their serorecognition of field-derived variant malaria peptides located in immunogenic epitopes. This first analysis was not designed to detect a minimum threshold of seroreactivity to epitopes needed to protect against clinical disease. Prospective longitudinal seroepidemiological studies measuring the association of seroreactivity to immunogenic epitopes and the risks of clinical malaria illness with homologous and heterologous parasites could demonstrate the basis for strain-specific clinical immunity. To facilitate this, next-generation diversity-reflecting protein and peptide microarrays will contain more variants of more antigens.38. Our earlier studies demonstrated that the AMA1 vaccine decreased the incidence of clinical malaria caused by parasites homologous to the vaccine strain with respect to the cluster 1 loop. Using a mutation scan method, we pinpointed a single amino acid (aa197) as a key determinant of the magnitude of strain-specific antibody responses to cluster 1 loop peptides after AMA1 vaccination. The relative importance of hypervariable locus position 197 was calculated using machine-learning random forest analysis9, and relative risk ratio models22, and was measured directly in this analysis. The effectiveness of a malaria vaccine displaying significant strain-specific efficacy would be limited by simultaneous or sequential infection with multiple parasite clones, which is common41.Effective malaria vaccines must do more than just elicit a strong immune response—the immune response must effectively inhibit parasite survival and replication, and ideally, the response should be strain-transcendingProtein and peptide arrays populated with field-derived variants of vaccine proteins are powerful tools to assess the immunogenicity of vaccine constructs and the strain-specificity of antibodies generated by vaccination. Peptide arrays are more expensive, and the analysis is labor-intensive, limiting their feasibility for routine sero-surveillance. Whole-protein arrays are relatively robust and inexpensive, and results can be reported within hours. While protein arrays are being developed for use in the field, like ELISA, they cannot easily characterize strain-specific or polyclonal antibody responses. The translation of these platforms into practical tools for seroepidemiology will require the thoughtful use of informative, strain-transcending biomarkers and economies of scale. Vaccine research may benefit from employing high-resolution peptide microarrays early in development to assess the strain-specificity of functional monoclonal antibodies and the serological responses to vaccination well before vaccines against malaria or other pathogens that use genetic diversity to evade the immune system are taken to the field for testing in humans.P. falciparum malaria transmission coincides with the rainy season between June and November. Entomological inoculation rates (EIR) range from a peak of 60 infected mosquito bites per person per month in September, to virtually no transmission during the dry season (December to May). The total annual entomological inoculation rate per person is estimated to be from 50 to 150 infected bites42. The vast majority (97%) of malaria infections are due to P. falciparum, with rare P. malariae, and P. ovale infections. Children under six years of age vary in their risk of clinical malaria episodes, ranging from none to more than four clinical episodes of malaria per year, depending on neighborhood of residence43.Bandiagara is a rural town with a population of approximately 14,000 inhabitants located in an area of seasonal malaria transmission in Mali. A, conducted in Bandiagara23. FMP2.1/AS02A is an AMA1-based vaccine consisting of the 449 amino acid AMA1 ectodomain (amino acids #83–531) of P. falciparum strain 3D7  and formulated with the proprietary AS02A adjuvant 45. In both studies, a licensed rabies vaccine (RabAvert) was administered to control groups.Sera were selected from two completed clinical trials of FMP2.1/AS0223. Participants who received the full dose of either the FMP2.1/AS02A (n = 20) or rabies control vaccine (n = 18) and had samples for all study time points were included in this study. Pre-vaccination sera were collected on the day of the first of three vaccinations, at the end of the malaria season . Sera were also collected and analyzed 90 days after the first vaccination in March 2005, when peak AMA1 antibody titers measured by IgG ELISA were observed in the AMA1-vaccinated cohort23. In addition, sera from the rabies-vaccinated adult cohort from the beginning (June 2005) and end (December 2005) of the malaria transmission season were probed on the array to detect differences in antibody reactivity after one malaria season. A subset of 10 control-vaccinated adults was randomly selected from the original 19 samples at the May time point and probed on the high-density peptide array.Adult serum samples were collected from Malians aged 18–55 years who had participated in a previously described phase I trial conducted from 2004 to 2005 in BandiagaraA) trial conducted in Bandiagara, Mali from 2007–200921. Children were vaccinated at the beginning of the malaria transmission season, May/June 2007; and peak AMA1 antibody titers were observed 90 days after first inoculation in September 2007. Post-season samples were collected from December 2007 through January 2008. Sera from 40 children in the AMA1 vaccine arm and 40 children in the rabies control vaccine arm were selected by random number generator (R Bioconductor) from the 200 originally randomized to each experimental arm in the vaccine trial.Pediatric serum samples were collected from children aged one to six years who participated in a previously-described phase II AMA1 vaccine  was used to identify single and predominant clone infections, and unique ama1 haplotypes were then amplified using a specific primer-linker sequence that facilitated cloning into a pXI plasmid vector. Cloning, transformation, and amplification protocols have been described46. Single-clone ama1 haplotype translation was ensured by use of a single colony amplification protocol25. Two hundred and sixty-three unique ama1 whole-protein sequences were observed among 412 sequences analyzed. Protein microarray printing, probing, data generation, and quality control protocols have been described46.Dried blood spots were collected during the phase II FMP2.1/AS0226. Conserved peptide sequences were printed only once on the array, with every fourth AMA1 peptide sequence printed in quadruplicate for quality control purposes. High-density peptide synthesis, sera probing and imaging protocols are described in Supplemental Methods.Three hundred and thirty-one full-length AMA1 sequences were derived from field samples (263 sequences) and publicly available sequences (68 sequences) and used to generate 16 amino acid long peptides which overlapped by 15 amino acids tiled across the length of AMA1ama1. For each child, sera from pre-season, 90 days post-vaccination, and post-season time points were probed on the peptide array. Ten Malian adults and five North American adults were selected from among those run on the whole-protein array by random number generator (R Bioconductor) to be run on the array as well.The peptide array was used to probe sera from 20 children selected from among those run on the whole-protein array. Ten AMA1-vaccinated children selected to be run on the peptide array were randomly selected from samples that met the following criteria: the participant did not have a malaria-positive sample between the date of first vaccination and 90 days post vaccination, they were also run on the whole-protein AMA1 array, and also had a statistically significant increase from pre-vaccination in AMA1 antibody titer in response to the vaccine as measured by ELISA and the AMA1 protein microarray. Ten control-vaccinated children were randomly selected from among those who had at least one dried blood spot positive for malaria during the first malaria transmission season, with parasites sequenced for An additional peptide array was created to perform a pilot mutation scan analysis on the cluster 1 loop (c1L): 3D7 AMA1 positions 190–206 (MSPMTLDEMRHFYKDNK), to assess the strain specificity of the serological response after AMA1-vaccination in the hypervariable c1L region. The array consisted of novel peptides fabricated such that each position in the 17 amino-acid-long c1L sequence was changed to each of the 20 canonical amino acids or a deletion, yielding 357 unique peptide sequences. Each unique sequence was replicated 7 times on the array. Among the children selected for the peptide array analysis, pre- and post-vaccination sera from six randomly-selected AMA1-vaccinated children were probed on the cluster 1 loop mutation scan array. These children did not have a malaria-positive blood sample within the first 90 days of follow-up to ensure responses were due to the AMA1 vaccine and not natural exposure to malaria parasites. Six children were selected due to the limited capacity of the array and the pilot nature of the experiment.7. Information regarding mAbs 479 A and 480 A obtained by MR4 is available online at https://www.mr4.org/. Monoclonal antibodies were probed at 1 mg/mL concentration and quantitated according to standard protein array protocols47.Monoclonal antibody 5A6 was generated by, and ordered from the Walter Reed Army Institute of Research. Monoclonal antibodies MRA-479A and MRA-480A were obtained from Malaria Research and Reference Reagent Resource Center (MR4). Protocols detailing monoclonal antibody 5A6 preparation and characterization were previously describedn-vitro transcription/translation (IVTT) negative controls to adjust for differences in seroreactivity to the IVTT reaction buffers and cell-free E. coli machinery. For peptides, raw MFI represented the magnitude of peptide seroreactivity. AMA1 peptides were synthesized in silico without an IVTT reaction and no adjustment for background seroreactivity was necessary. The mean seroreactivity to AMA1 domains and structural loops was calculated by taking the mean seroreactivity to 16-mer peptides whose N-terminal amino acid, (the end unlinked to the substrate linker sequence) falls within the range identified as part of each structure or domain.The magnitude of seroreactivity to whole-protein AMA1 variants was calculated by scaling the raw median fluorescence intensity (MFI) of all probes on the protein array to 15 empty vector iAll matched-pair analyses were done using the Wilcoxon sign-rank test. Group-wise, non-matched comparisons were conducted using the Mann Whitney test. Kruskal-Wallis non-parametric ANOVA was used to compare more than two groups. All statistical analyses were performed with R Project for Statistical Computing, Version 3.2.3.Strain-specific serological profiles were generated by matching 16-mer peptides to the sequence of the infecting parasite (homologous) and the reference strain 3D7 on which the vaccine is based. Seroreactivity to peptides homologous to the infecting parasite strain was then averaged by amino acid position among all children who had a clinical malaria episode during the first 90 days of follow up. Seroreactivity to peptides matching strain 3D7 was also averaged as a reference for comparison.S for sample i at position j, mutation k:Normalized signal was calculated for each sample by amino acid position such that if all mutations at a position produced equal signal, the normalized signal for each mutation at that position will equal a value of 1. Normalized signal was calculated by taking signal 48 were calculated with: stats::p.adjust.A neutral amino acid position, as defined by the substitution of all 20 potential amino acids and a deletion while still resulting in an equal signal with respect to the original amino acid, will yield a normalized signal of 1for all mutant sequences at that position. T-tests were performed at each position for each mutation to determine whether the normalized signals in the sample cohort were significantly greater than 1 after vaccination. Benjamini-Hochberg adjusted p-values were calculated resulting in 23 individual mutations, where association was reported with an adjusted p-value less than 0.05. Statistical analysis was performed in R version 3.0.2. T-tests were performed with: stats::t.test. False discovery rate adjusted p-valuesSupplementary information.Supplementary Data file S1.Supplementary Data file S2.


Plasmodium falciparum in SCD patients can trigger painful vaso-occlusive crisis, increase the severity of anaemia, and contribute to early childhood mortality.Patients with sickle cell disease (SCD), an inherited haemoglobinopathy, have increased risk of malaria, at least in part due to impaired splenic function. Infection with A 17 year-old Tanzanian male with known SCD was admitted to Muhimbili National Hospital, a tertiary referral centre in Dar-es-Salaam, following an attack of malaria. From 2004 to 2007 the patient had lived in USA, and from 2010 to 2016 in France where, on account of hypersplenism and episodes of splenic sequestrations, in 2014 the spleen was removed. After appropriate clinical and laboratory assessment the patient was re-started on hydroxyurea; and anti-malarial-prophylaxis with proguanil was instituted. The patient has remained well and malaria-free for the following 15 months.SCD patients are highly vulnerable to malaria infection, and impaired splenic function is a feature of SCD patients, even in those who still anatomically have a spleen. This patient had a surgical splenectomy and, in addition, had probably lost some of the acquired malaria-immunity by having lived for several years in malaria-free areas. This patient is a compelling reminder that long-term anti-malarial prophylaxis should be offered to all patients with SCD who live in malaria-endemic areas. Sickle cell disease (SCD) is an autosomal recessive disorder characterized by chronic haemolytic anaemia and painful vaso-occlusive crises. SCD is common in sub-Saharan Africa, with a birth prevalence that in some areas reaches 2% , and whePlasmodium falciparum-infected erythrocytes [SCD patients also experience increased susceptibility to severe infections, including malaria , 4. Thishrocytes . On the hrocytes , 4, 7.Splenic dysfunction may be responsible for increased susceptibility of SCD patients to malaria as well as to other infections , 9, as rIn view of the higher malaria mortality rate in SCD patients, although it is questionable whether anti-malarial prophylaxis reduces the frequency of vaso-occlusive crises , this prThis is a report of a patient with SCD who was asplenic, who had lived for several years in malaria-free environment and who, upon return to Tanzania, did not initially receive anti-malarial prophylaxis and suffered recurrently from malaria.Plasmodium falciparum malaria infection was diagnosed upon microscopic examination of blood slides. The patient was treated with artesunate injection followed by quinine tablets. A similar episode, with confirmed P. falciparum infection, had occurred some weeks earlier, when the patient had been treated with artemether-lumefantrine tablets.A 17 year-old Tanzanian male with known SCD came in May 2018 to the Sickle Cell Clinic at the Muhimbili National Hospital (MNH) in Dar-es-Salaam, Tanzania with a complaint of loss of appetite and nausea. The day before the patient had been discharged from a local hospital in Dar-es-Salaam where, having been admitted 5 days previously because of fever, nausea, loss of appetite, generalized body pain and weakness. The medical history showed that the patient had been diagnosed with SCD at the age of 2 years. At the age of 5 years when living in the USA, the patient was started on hydroxyurea. In 2007, he returned to Tanzania where, in 2008, he had an episode of malaria. In 2010, the patient moved to France where further investigations revealed that his SCD was sickle beta thalassaemia, and that he was also homozygous for alpha-thalassaemia. The beta thalassaemia mutation was identified as IVS1nt5G > C. Deficiencies of coagulation factors VII and X were also detected. His steady-state Hb level was 10–11 g/dL; however, he required blood transfusion on 9 occasions because of recurrent exacerbations of anaemia, attributed to hypersplenism and recurrent splenic sequestration. Therefore in 2014 he had a laparoscopic splenectomy. The spleen weighed 428 g and histology showed marked congestion without haemosiderosis or other abnormalities.In 2016, the patient returned to Tanzania where he now attends secondary school. In 2017 the patient was feeling so well that he stopped taking hydroxyurea, his steady-state haemoglobin being 9–10 g/dL, and his only medication was folic acid (5 mg/day). However, recently he has had several bone pain crises.On admission to MNH in May 2018 clinical examination did not reveal any specific findings. There was no severe pain and no evidence of infection. The patient’s Hb was 7.3 g/dL and a malaria test was negative. The patient continued on folic acid, hydroxyurea (500 mg/day) was re-introduced, and anti-malarial prophylaxis with proguanil (100 mg/day) was started. On monthly follow-up visits from June 2018 to September 2019 the patient has been generally well, except for occasional pain that responded to paracetamol, diclofenac or ibuprophen. Laboratory findings  The patient had sickle beta thalassemia, a well known form of SCD; (ii) he was asplenic; and (iii) after being born and having lived in Tanzania for the first 4 years of his life, he lived for a total of 8 years in malaria-free countries (USA and France), whereby whatever immunity he had previously developed may have faded. He had at least two documented episodes of malaria infection prior to his splenectomy, and at least two after splenectomy. In this patient, therefore, the spleen was removed for good clinical reasons, and the procedure was recommended only after a balanced judgment of pros and cons. One goal was to reduce substantially the rate of blood transfusion, and this was achieved. As for the implications of being asplenic, it must be accepted that living in a malaria-endemic region is an added risk.Malaria episodes in this patient have been treated promptly: fortunately, because in a splenectomized SCD patient it might rapidly become life-threatening, a decision was made to institute anti-malarial chemoprophylaxis . Officiafalciparum malaria, an exogenous extra threat that is preventable.This patient was a living reminder that if SCD cannot be cured, then at least patients with SCD should be protected from


Malaria remains a serious public health problem globally. As the elimination of indigenous malaria continues in China, imported malaria has gradually become a major health hazard. Well-timed and accurate diagnoses could support the timely implementation of therapeutic schedules, reveal the prevalence of imported malaria and avoid transmission of the disease.Plasmodium species by using 18S rRNA-based nested PCR and Taqman probe-based real-time PCR. The results of the microscopy and the two molecular diagnostic methods were analysed. Importation origins were traced by country, and the prevalence of Plasmodium species was analysed by year.Blood samples were collected in Wuhan, China, from August 2011 to December 2018. All patients accepted microscopy and rapid diagnosis test (RDT) examinations. Subsequently, each of the positive or suspected positive cases was tested for four human-infectious Plasmodium falciparum positive, and 1 suspected case, were collected and reanalysed. After application of the two molecular methods and sequencing, 291 cases including 245 P. falciparum, 15 Plasmodium vivax, 20 Plasmodium ovale, 6 Plasmodium malariae and 5 mixed infections  were confirmed. These patients had returned from Africa (95.53%) and Asia (4.47%). Although the prevalence displayed a small-scale fluctuation, the overall trend of the imported cases increased yearly.A total of 296 blood samples, including 288 that were microscopy and RDT positive, 7 RDT and These results emphasize the necessity of combined utilization of the four tools for malaria diagnosis in clinic and in field surveys of potential risk regions worldwide including Wuhan. Malaria remains a serious threat to public health around the world, with an estimated 228 million cases and 405,000 deaths in 2018 worldwide . In ChinPlasmodium species causing malaria in humans, including Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. Different Plasmodium species infections can cause dissimilar disease characteristics. Amongst them, P. falciparum is responsible for most of the morbidity and mortality of humans in sub-Saharan Africa, and other malarious tropical areas around the world [Plasmodium falciparum can also increase the risk of severe malaria and adverse complications in pregnant women, such as maternal death, miscarriage, stillbirth and neonatal death. If patients fail to get diagnosis and treated within 24 h after the onset of falciparum malaria, it may be fatal [Plasmodium vivax is generally the culprit for the majority of malaria cases in areas with tropical and temperate climates and is considered to be a neglected tropical disease [P. vivax malaria cases have been reported in endemic areas of (sub) tropical countries. For P. vivax, a dormant form named hypnozoite, could stay in the liver and may lead to the malaria relapses several months or even years later [Plasmodium ovale is mainly prevalent in Africa and Asia, particularly in West Africa [Plasmodium ovale seldom leads to severe malaria among people in endemic regions, but can cause severe malaria disease in naive visitors [P. ovale curtisi and P. ovale wallikeri [Plasmodium ovale curtisi has a significantly longer latency duration than P. o. wallikeri [P. vivax, P. ovale can also form hypnozoites in the host liver, which makes the prevention and treatment of both vivax and ovale malaria more difficult [Plasmodium malariae cases have been reported worldwide and are particularly prevalent in West Africa. Plasmodium malariae usually causes the mildest infections and very rarely has life-threatening results. However, it may cause splenomegaly or renal damage during chronic infection [Plasmodium knowlesi was initially found in SE Asia in wild macaques, but it can also infect humans [Plasmodium species commonly happen, especially in endemic regions [P. vivax, P. ovale and P. malariae as well as mixed infections is frequently underestimated [Plasmodium infections should be treated with the corresponding chemoprophylaxis and appropriate treatment principles [Plasmodium species infection is meaningful.There are five primary he world , 7. Popube fatal . Plasmod disease , 10. Sevrs later . Plasmodvisitors , 12. It allikeri . Plasmodallikeri , 15. Likifficult . Plasmodt humans . Althougt humans , 17, 18. regions . Howeverstimated –22. MostPlasmodium species are frequently missed [P. ovale  cannot be distinguished by microscopy. Misidentification may also happen between different species [Plasmodium-specific antigens in blood samples such as histidine-rich protein 2 (HRP2) and lactate dehydrogenase (LDH) [Plasmodium species infection [Plasmodium infection, but also allow for accurate species identification by primer design should be used. Nested PCR with two rounds of amplification can improve the detection sensitivity. Sequencing of products from nested PCR represents a further reliable guarantee for species identification [As it is inexpensive and has a relatively high sensitivity for species recognition and even parasite density quantification, microscopic examination of thick and thin blood smear is traditionally regarded as the gold standard for malaria detection , 24. Howy missed . The two species . All of se (LDH) . RDT offnfection . Furthernfection –34. Therfication , 36. Thefication , 38.Plasmodium species involved in all imported malaria cases in Wuhan by using microscopy, RDT, nested PCR and real-time PCR. The performances of these methods in malaria diagnosis were assessed to supply more reference values for their future use. Analysis of their diagnostic results revealed the prevalence and characteristics of imported malaria in Wuhan, China.Using suitable and effective methods to timely identify imported malaria is essential for successfully completing the target assessment task for malaria elimination in China, Africa and SE Asia. Therefore, the present study aimed to confirm the Blood samples from clinically suspected patients with symptoms of malaria were cumulatively collected from the Center for Disease Prevention and Control (CDC) of Wuhan, China, from August 2011 to December 2018. Approximately 2 to 5 ml blood was drawn from each patient for microscopy and RDT testing and 400 μl of each sample was stored on 3 MM Whatman filter paper for molecular verification. Ethical approval for this study was obtained from the Medical Ethics Committee of the Hubei University of Medicine and Wuhan CDC. Informed consent was provided by all participating individuals.Plasmodium detected in 200 high-power fields in the thin blood films and no parasites for every 1000 white blood cells were considered as negative [The blood samples were first subjected to One Step Malaria HRP2/pLDH (P.f/Pan)  detection. Subsequently, thick and thin peripheral blood smears were made by the standard method . Giemsa negative , 41.Genomic DNA (gDNA) from the microscopic-positive or suspected positive blood samples was extracted using a TIANamp blood DNA kit . Briefly, 6 mm × 6 mm blood spot  was treated following the manufacturer’s instruction and finally dissolved in 50 μl elution buffer. After quantification with Gene 5, the purified gDNA was packed as aliquots and stored at − 20 °C until further use.P. falciparum, P. vivax, P. ovale  and P. malariae, so they could be distinguished with real-time PCR. The preparation of primers and Taqman probes was according to previous documents [Probes were designed for the parasite species, including ocuments , 42  gene were synthesized and used as previously described [2+ and 2 × PCR buffer), 1 μl of each primer  and 1 μl DNA template in a final total volume of 25 μl supplemented with ultrapure water. The amplification was carried out using the following conditions: initial denaturation at 95 °C for 3 min, 30 repeated cycles at 95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min, followed by a final extension at 72 °C for 5 min. A 1670 bp PCR product was obtained from each sample after the primary amplification, which was then used as the DNA template for the secondary amplification. The reaction system and conditions in the second round were similar to the first round with minor modifications. Briefly, the annealing temperature was adjusted to 56 °C, and the extension time was changed to 30 s, considering the amplification products were shorter in the second reaction. All the products from the secondary amplification were subjected to 1% agarose gel electrophoresis and judged by their band sizes. Products of the nested PCR were used for sequencing if divergent results occurred among the microscopy and the two molecular methods.The above samples were further subjected to nested PCR. For nested PCR, the classical primers targeting the escribed , 44  were obtained from online calculator named vassarstats [To calculate the diagnostic sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), disease prevalence (DP) and accuracy of four sarstats . BrieflyP. falciparum, 17 P. vivax, 21 P. ovale, 6 P. malariae and 1 coinfection  were observed. Seven out of the remaining 8 cases (296 minus 288) were microscopy negative but RDT positive , 26 (10.70%), 34 , 30 (12.35%), 96 (39.51%) and 50 (20.58%), respectively. For P. vivax, parasitaemia ranged from 100 to 30,000 parasite/μl and the mean parasite density was 4600 parasite/μl (95% CI 989–8211). For P. ovale, parasitaemia ranged from 500 to 10,000 parasite/μl and the mean parasite density was 2610 parasite/μl (95% CI 1348–3871). Of which, 23.53% (4/17) of P. vivax and 19.05% (4/21) of P. ovale had parasite densities ≤ 500 parasite/μl. All of the 6 P. malariae cases had parasite densities from 800 to 4000 parasite/μl and the one mixed infection was 50,000 parasite/μl  or in the microscopy-negative group. For the 12 cases that were not detected by real-time PCR, they were all caused by P. falciparum. Their parasite density distributions were highly in accordance with those in the nested PCR detection, including 2 cases in the Very low group, 5 cases in the Low group, 1 case in the Low-middle group and 4 cases in the microscopy-negative group. Additionally, both molecular tools revealed one P. falciparum plus P. ovale mixed infection case among the microscopy-positive P. falciparum samples. The one suspected case negative by both microscopy and RDT was proven to be P. falciparum infection.These samples were confirmed via both nested PCR and real-time PCR. After reconfirmation by nested PCR  P. vivax, 21 (7.09%) P. ovale, 6 (2.03%) P. malariae, 1 (0.34%) mixed infection  and 8 (2.70%) negative cases according to microscopic observations. Based on the nested PCR, 245 (82.77%), 15 (5.07%), 20 (6.76%) and 6 (2.03%) cases were identified as single infection by P. falciparum, P. vivax, P. ovale and P. malariae, respectively, whereas dual species mixed infections included 3 (1.01%) cases of P. falciparum + P. ovale, 2 (0.68%) cases of P. vivax + P. ovale and 5 (1.69%) negative cases. Real-time PCR revealed a few minor discrepant results, including 238 (80.41%) P. falciparum, 15 (5.07%) P. vivax, 22 (7.43%) P. ovale, 6 (2.03%) P. malariae, 1 (0.34%) mixed infections of P. falciparum + P. ovale, 2 (0.68%) cases of P. vivax + P. ovale and 12 (4.05%) negative cases. No triple or quadruple infection were detected in these samples.As shown in Fig. Plasmodium species as well as the following performance assessment of the diagnostic methods. As shown in Table P. falciparum, P. vivax, P. ovale and P. malariae were 96.77%, 100%, 88.00% and 100.00%, respectively. Specificities for P. falciparum diagnosis were 91.67% and 100.00% for the other three species. The probabilities of identification of P. falciparum, P. vivax, P. ovale and P. malariae by microscopy from the predicted positive (PPV) and not identification from the predictive negative (NPV) samples were 98.36% and 84.62% , 100.00% and 100.00% (P. vivax), 100.00% and 98.91% , 100.00% and 100.00% , respectively. For the detection of P. falciparum by real-time PCR, sensitivity, specificity, PPV and NPV were 96.37%, 100%, 100% and 84.21%, respectively. These four assessment indexes were all 100% for the other three species diagnosed by real-time PCR.Nested PCR was appointed as a reference for the diagnostic summarizing of the P. falciparum (83.78%) infection was the most prevalent, followed by P. ovale (8.45%), P. vivax (5.74%) and P. malariae (2.03%) among the imported malaria cases in Wuhan, China. Since RDT could only distinguish falciparum and non-falciparum Plasmodium species infection, RDT was not involved in the methodological evaluation of these samples.The results also revealed that P. falciparum was in strong accordance with above general tendency, including 102 cases from West Africa, 55 from Central Africa, 55 from Southern Africa, 27 from East Africa and 6 from SE Asia. Amongst the P. vivax patients, 8 returned from East Africa. The remainders all returned from SE Asia (3 cases) and South Asia (4 cases), respectively. The P. ovale patients returned from West Africa (10/20), Central Africa (8/20) and Southern Africa (2/20). Only 6 P. malariae infected patients were involved, and three returned from Southern Africa, two from Central Africa and one from East Africa. The five mixed infection patients also returned from the same three regions as the P. malariae infection group.Tracing the origin of the imported malaria patients demonstrated the patients returned from 28 countries of Africa and 4 countries of Asia Fig. a. Of theP. falciparum throughout the surveyed period. However, it is worth mentioning no consistent increasing trends in the prevalence of P. falciparum parasites infection  was detected yearly. Other Plasmodium species, including P. vivax, P. ovale and P. malariae single infection or mixed infection, were not identified until 2014. In the next 5 years (2014–2018), patients infected with P. vivax (15 cases), P. ovale (20 cases), P. malariae (6 cases) and mixed parasite species (5 cases) were seen. In this period (2014–2018), the total imported non-falciparum cases showed no increasing trend in prevalence by year . Neither P. vivax  nor P. ovale  significantly increased by year.From 2011 to 2016 Fig. b, the nuIn 2020, China hopes that national malaria elimination will become a real possibility based on effective control measures . AlthougP. falciparum was the dominant imported species. Analysis of the performance of the diagnostic methods involved also revealed some limitations in their usage. Of the 243 P. falciparum cases identified by microscopy, one case later proved to be infected with P. falciparum plus P. ovale by both molecular tests. Two microscopy-positive P. vivax cases were also found to be cases of P. vivax plus P. ovale. Likewise, two patients with single P. ovale infection by microscopy were proven as P. ovale plus P. falciparum infection by nested PCR. In previous studies, P. ovale was also frequently reported to be involved in mixed infection with other Plasmodium species [P. o. curtisi and P. o. wallikeri in 20 single P. ovale infection cases, which were unable to be distinguished by microscopy, also highlighted the advantages of molecular tools in species identification. Their diagnosis will offer a clue for the precise treatment of malaria patients. Subsequently, nested PCR also proved 6 cases as P. falciparum infection, which included 5 microscopy-negative but RDT-positive cases and one double-negative case. This was probably because the level of parasitaemia was too low to be observed in these cases. As reported in previous studies [Plasmodium, but the threshold, 50 parasites/μl, is rarely noticeable unless under optimum conditions. This means that it is quite difficult for observers to accurately identify the species with relatively low parasitaemia, especially in mixed infections.In the current study, several discrepant results were obtained by the four different diagnostic methods, which ultimately revised 5 cases from single to mixed infection and 6 cases from negative to positive. The confirmed data revealed that 95.5% of the imported malaria patients had returned from Africa, and  species , 46, 47. species . The ide studies , 50, micP. falciparum identification. However, the specificity of microscopy was only 91.67%, lower than the 100% of rea1-time PCR detection. By designing complementary probes to target gene sequences, the Taqman probe is another guarantee besides the primers to enhance the specificity and avoid false-positive results [P. falciparum detection was approximately 85% in both microscopy and real-time PCR detection, indicating a relatively high false-negative rate of approximately 15% for these two methods. A false-negative rate as high as 19.4% for microscopy was reported in a previous study [P. falciparum cases consisting of 7 samples with low parasite density (0–500 parasites/μl) and 2 mixed infection samples. In a previous study, the same real-time PCR primers and probes were assessed [P. falciparum detection was 100%. This indicated that diagnostic performance may vary with the reaction conditions, including the DNA quality, reaction system and even the performance of the laboratory technicians, remaining us of the significance for optimization of the reaction system. For P. ovale, the sensitivity (88.00%) of microscopic identification was relatively low, and the NPV for P. ovale was 98.91%. Two of the 3 P. ovale cases that failed to be detected by microscopy were proved to be P. ovale plus P. vivax mixed infections. This may be due to the similar morphology between P. ovale and P. vivax [P. vivax, P. malariae and P. ovale detection, revealing the high consistency between the two molecular tools in the identification of these three species. Plasmodium malariae could be accurately diagnosed by all three methods. This may be because only 6 P. malariae cases were involved in the study. The primers for real-time PCR used in this study have been used in several previous studies [Plasmodium infection failed to be diagnosed. This could cause a misdiagnosis, delayed treatment of malaria and even lead to a life-threatening problem, especially for P. falciparum infection. Beyond the individual consequences, false-negative patients may also spread malaria and produce resistance to anti-malarial drugs if given incorrect treatment due to the lack of an exact diagnosis [In the analysis of sensitivity, specificity, PPV and NPV, nested PCR-based detection was chosen as the reference standard. Actually, nested PCR is commonly selected as a reference method , 52. By  results . The NPVus study . In anotus study . These iassessed . AlthougP. vivax ; as a re studies , 56. How studies , 58. A fiagnosis .Plasmodium species can then be confirmed by molecular methods, including nested PCR and real-time PCR.Although nested PCR showed the highest sensitivity and real-time PCR had the best specificity, the role of microscopy and RDT is not controversial. Use of the two molecular methods relies on DNA extraction from blood samples. If errors happen during the extraction, a false-negative would probably occur. For example, in a previous study, 10 malaria cases failed to be identified by molecular methods due to problems with the nucleic acid extraction, and the reasons were only identified after repeated experiments . TherefoPlasmodium infection [P. falciparum were similar to other studies [P. falciparum should be highly considered in Wuhan. Over the years (from 2014 to 2018), the Plasmodium species leading to imported malaria were becoming diversified, just as in other provinces of China, including Shandong [P. vivax/P. ovale cases showed no significantly increasing trend by year, but more attention should be focused on this increasing Plasmodium species diversity. The increase in imported P. vivax and P. ovale cases may cause the re-introduction of malaria into regions without cases [Plasmodium malariae can survive in humans for many years and keep infectivity of its vector at a truly low parasitaemia load [Plasmodium infection worldwide, even in China.The origin of infection of the patients was consistent with a previous report referring to the epidemiologic features of imported malaria in China . In the nfection . Additionfection . Workers studies , 62. TheShandong , ZhejianShandong  and ChonShandong . In thisut cases , 66. Plamia load . The shaAdditionally, results in the present study showed no significant seasonal correlation P = 0.759) by the Pearson correlation analysis among the imported malaria cases during the study period. One small peak of the seasonal index  appeared9 by the Plasmodium species at the destination and the conditions of the travellers , different chemoprophylactic schedules should be advised by the local government according to the guide on international travel and health published by the WHO. Only a combination of prevention and treatment can lead to the achievement of the goal of malaria elimination around the world.Indeed, besides an early and precise diagnosis for proper treatment, some preventative measures should also be emphasized from two major aspects before people leave for an epidemic area: (1) Prevention of mosquito bites. Travellers could protect themselves by using insect repellents, insecticide-treated nets, aerosol sprays and protective clothing. (2) Proper usage of chemoprophylactic anti-malarial drugs. Anti-malarial prophylactic regimens may not provide complete protection, but proper chemoprophylaxis could significantly lower the risk of fatal malaria. According to the prevalence of Plasmodium species and two subspecies of P. ovale was assessed. The results highlighted, once again, that PCR-based methods are irreplaceable for exact species determination of imported malaria cases, especially for mixed infections. Therefore, the combined use of the four methods should be emphasized, if conditions permit. These data are a very important epidemiological resource and could supply guidance for the surveillance, prevention and timely treatment of imported malaria in Wuhan, China. The guiding effect is also significant for epidemic areas, as shown in the origin distribution survey, and for countries at potential high risk of importing malaria worldwide.The efficiency of 18S ssrRNA-based nested PCR and real-time PCR in the differentiation of four


To test the hypothesis that malaria might cause ID, we used sickle cell trait , a genetic variant that confers specific protection against malaria2, as an instrumental variable in Mendelian randomization analyses. HbAS was associated with a 30% reduction in ID among children living in malaria-endemic countries in Africa , but not among individuals living in malaria-free areas . Genetically predicted malaria risk was associated with an odds ratio of 2.65 for ID per unit increase in the log incidence rate of malaria. This suggests that an intervention that halves the risk of malaria episodes would reduce the prevalence of ID in African children by 49%.Malaria and iron deficiency (ID) are common and interrelated public health problems in African children. Observational data suggest that interrupting malaria transmission reduces the prevalence of ID Similarly, ID is also common and accounts for over 60% of anemia cases 5. ID is associated with impaired cognitive development 6 and is a leading cause of years lived with disability in African children7. ID may be caused by a number of factors, including diets low in available iron, dietary iron inhibitors, such as polyphenols and phytates, and helminth infections. Iron supplementation, via tablets or syrups and micronutrient powders, are the primary interventions to manage ID and anemia in children. However, there are long-standing concerns regarding the safety and efficacy of iron supplements in malaria-endemic countries, where they may predispose individuals to malaria and other infections8 and be poorly absorbed10. New strategies are needed for the management of ID.Malaria and ID are important co-existing public health problems across sub-Saharan Africa, and their relationship is complex and incompletely understood. Malaria transmission is persistent and widespread across sub-Saharan Africa with an estimated parasite prevalence of 24%  compared to children lacking such protection.Malaria is known to cause anemia via various mechanisms, including the destruction of parasitized and non-parasitized erythrocytes. This process affects hemoglobin concentrations, but the iron released from destroyed erythrocytes is not lost from the body and can be recycled. Little is known about whether malaria might be a cause of ID in African childrenPlasmodium falciparum malarian=11 of 845). These studies are described in detail in the Methods. We also considered using other polymorphisms, including glucose-6-phosphate dehydrogenase (G6PD) A deficiency and α-thalassemia, as instrumental variables, but, in meta-analyses of published literature (-1 or <30µgl-1 in the presence of inflammation (defined as C-reactive protein (CRP) >5mgl-1 or α1-antichymotrypsin (ACT) >0.6gl-1 or α1-acid glycoprotein (AGP) >1gl-1) in children <5 years old or <15 μgl-1 in children <5 years old-1 in African American adults and Nairobi school children-1 in children aged <5 years, <115gl-1 in children ≥5 years, <120gl-1 in women and <130gdl-1 in men and ID anemia (IDA) as the presence of ID and anemia17. The characteristics of the study populations are summarized inWe used sickle cell trait , a genetic variant that confers specific protection against all forms of clinical terature , the proI2 = 0.0%, 18, we further adjusted for the effect of α-thalassemia, but this did not alter the effect of HbAS on risk of ID  , suggesting that the genetically predicted risk of uncomplicated malaria is associated with 2.65-fold higher odds of ID per unit increase in the log incidence rate of malaria. We then calculated the effect on ID of reducing malaria incidence by half. We obtained this by multiplying the causal log odds (0.97) by the natural logarithm of 0.5 and then exponentiated the result20. We observed an OR of 0.51, suggesting that reducing the incidence of uncomplicated malaria by half would reduce ID by 49%. In sum, our findings suggest that malaria is an important cause of ID and that malaria control may be an effective strategy for addressing ID among children living in sub-Saharan Africa.We then estimated the causal effect of malaria on ID using HbAS as an instrumental variable for malaria in a two-sample Mendelian randomization analysisio (IRR) . Overallio (IRR) , and theAS on ID  to log iAS on ID  with a M-1 and 100.28mgl-1,respectively) compared to levels in those without malaria (1.15mgl-1). Carriage of HbAS was associated with a 25% reduction in inflammation  from children across the African continent and from African Americans, we provide evidence that malaria increases the risk of ID in African children . The public health benefits of malaria control in terms of reducing ID depend on the efficacy of the specific interventions used. Our data suggest that an intervention that halves the incidence of malaria would reduce ID by 49%. In a small study of Ivorian children, the prevalence of ID was reduced by half following treatment for afebrile malaria9, suggesting that the predicted benefits from our analysis may be translatable to the real world. Studies in malaria-endemic areas have focused on the potential for iron status to influence the risk of malaria infection. These studies have led to questioning whether iron supplementation can be safely given in the management of ID in malaria-endemic areas29. However, our findings suggest that malaria itself may be causing ID in African children. Other infections may similarly cause ID; for example, respiratory infections were associated with hepcidin-mediated ID in Gambian children30. The management of ID has traditionally involved iron supplementation. However, in addition to long-standing concerns regarding the safety of iron supplementation in Africa, there are also concerns regarding effects on the gut microbiome31 and lack of efficacy in areas of high infectious burden where chronically raised hepcidin levels may inhibit iron absorption10. Interventions that reduce malaria would allow iron to be more effectively absorbed from dietary sources. We recommend that strategies to prevent and treat malaria and other infections should be an integral part of programs to control ID in African children. Future research should confirm these findings by conducting trials of malaria control, for example, intermittent preventative treatment trials of malaria to evaluate the effect of malaria control on iron status.In summary, using large-scale data , Tanzania, The Gambia, Cameroon and Uganda) living in malaria-endemic countries. Four of these studies  were cross-sectional studies, two were longitudinal (in Tanzania and Kilifi Kenya), two were RCT (in Burkina Faso and Uganda), one was a cluster RCT (in Western Kenya), and one was a cluster survey (in Cameroon). The study also included three cohorts living in countries with no malaria exposure, African American adults from the longitudinal JHS, children from Soweto, South Africa and a survey of school children in Nairobi, Kenya. These studies are described below.32. The MMS included all children aged 6–59 months from randomly selected clusters and households. Details of the study design are available elsewhere32. Briefly, whole blood collected in tubes containing EDTA was used to test for malaria using a rapid diagnostic test (RDT) , Alere) and to measure hemoglobin concentrations using the HemoCue 301 system (HemoCue America). Levels of serum ferritin, CRP and AGP were measured using sandwich ELISA (VitMin Laboratory)33. Genotyping of sickle cell trait and α-thalassemia was performed using PCR as previously described32.The 2015–2016 Malawi Micronutrient Survey (MMS) was conducted as part of the Malawi Demographic and Health Survey. This cross-sectional survey aimed to determine the prevalence of anemia, micronutrient deficiencies (iron and vitamin A), infections and hemoglobinopathies34. Children aged 6–59 months were recruited from three strata  in Ghana, and random selection was performed in each stratum. Blood sampling and anthropometry were conducted during the survey. Malaria testing was performed using RDT ; hemoglobin concentrations were measured using HemoCue 301 AB). Sandwich ELISA33 was used to measure serum ferritin, CRP and AGP concentrations. DNA was extracted from blood pellets and used to type sickle cell trait and α-thalassemia using PCR36.This study was part of the 2017 Ghana Micronutrient Survey. Details of the cross-sectional study design and ethical approvals are presented in ref.37. Infants between the ages of 6 and 18 months living in the Banfora region of Burkina Faso were recruited into a phase 1–2b clinical trial to test the safety, immunogenicity and efficacy of an experimental heterologous viral-vectored prime-boost liver-stage malaria vaccine37. Serum ferritin , hepcidin (DRG Hepcidin 25 (bioactive) High Sensitive ELISA kit ), CRP , hemoglobin  and malaria parasitemia (Giemsa-stained thick and thin blood films) were measured at a single time point. Genotyping of sickle cell trait in the VaccGene study is described below.This was part of the VaccGene study, which aimed to identify genetic variants associated with differential response to vaccination in infancy but with ethical approval to undertake analyses to examine the effect of iron status on infection susceptibility. Details of the study design are described elsewhere38. Samples collected from the environmental enteropathy endline survey were used. Venous blood samples were used to test for malaria parasitemia using RDT , Alere) and hemoglobin concentrations (HemoCue Hb 301). Serum ferritin and CRP levels were assayed using sandwich ELISA33. Serum hepcidin-25 levels were quantified by using a competitive ELISA kit (PenLabs). Genotyping of sickle hemoglobin types and α-thalassemia was conducted using PCR36.Children were recruited from rural villages in Bungoma, Kakamega and Vihiga counties in western Kenya, using a cluster study design as part of the water, sanitation and handwashing benefits RCT . Details of the study design were published elsewhere39. Venous blood samples were used to test for malaria parasitemia using RDT . Serum ferritin, CRP and AGP levels were assayed using sandwich ELISA33. Hemoglobin typing for sickle cell trait was conducted using pyrosequencing, while PCR was used to detect α-thalassemia as described elsewhere39.This study used data collected during a nutrition cross-sectional survey of mothers and their children aged 6–59 months in rural Sud Kivu and Kongo Central provinces in the DRC as described elsewhere40. Within this cohort, children were followed to 8 years of age with weekly follow-ups and annual cross-sectional surveys during which anthropometry measurements were made and blood samples were collected. Serum ferritin , hepcidin (DRG Hepcidin 25 (bioactive) High Sensitive ELISA kit ), CRP , hemoglobin  and malaria parasitemia (Giemsa-stained thick and thin blood films) were measured from blood samples collected at a single cross-sectional survey based on the availability of plasma samples archived at 80°C. Genotyping of hemoglobin types and α-thalassemia was conducted by PCR36 using DNA extracted with the Qiagen DNA Blood Mini kit (Qiagen).This was an ongoing rolling longitudinal study designed to evaluate immunity to malaria in children and is described elsewhere41. Blood samples were collected at 3, 6 and 12 months of age and then once every 6 months in years 2 and 341. In this study, we used data from samples collected at a single time point, each child’s oldest time point, as older children were likely to have experienced more malaria episodes. The presence of P. falciparum parasitemia was determined using Giemsa-stained thick blood smears, while hemoglobin levels were measured using an impedance-based analyzer (Abbott Cell Dyn 1200). Plasma ferritin and CRP levels were assayed using a multiplex bead-based platform (Bio-Rad) and custom assay kits, and hemoglobin was typed by electrophoresis (Helena Laboratories)41. Genotyping for α-thalassemia was conducted as described by Chong et al.Children were enrolled at delivery into the MOMS Project longitudinal birth cohort at Muheza District Hospital in northeastern Tanzania between 2002 and 2006. Children were assessed for malaria parasitemia every 2 weeks during infancy and monthly thereafter, as well as at the time of any illness. Singleton children without evidence of human immunodeficiency virus in themselves or in their mothers during follow-up were included in studies of iron status and risk of malaria infection as described elsewhere42. We used cross-sectional data collected at the start of the malaria season. All children had a clinical examination, anthropometric measurements and a 3-d course of mebendazole for possible hookworm infection. A blood sample was collected for complete blood count, observations with a malaria slide, measurements of ferritin (Microparticle Enzyme Immunoassay (Abbott Architect)), hepcidin (Hepcidin-25 (human) EIA Kit (Bachem) and ACT  levels and DNA extraction. Children with a temperature >37.5°C had a malaria blood film, appropriate clinical treatment and a blood sample 2 weeks later after recovery from illness. Genotyping of sickle cell was performed on amplified DNA as detailed elsewhere42. The Gambian Bachem hepcidin values were harmonized by converting to the old DRG hepcidin assay values +1.633) and then to the new High Sensitive DRG hepcidin assay values -3.24) as previously validated43.All children aged 2–6 years old were recruited from ten rural villages in the West Kiang region of The Gambia during the malaria season (July to August 2001)44. Venous blood samples were collected for malaria testing using RDT  and hemoglobin measurement using a photometer (HemoCue). Plasma ferritin, CRP and AGP levels were assayed by ELISA33. Hemoglobin genotypes were determined by HPLC using an ultra-Resolution Variants Analyzer (Trinity Biotech), while α-thalassemia type was determined by PCRChildren 12–59 months of age were recruited to a cluster survey that aimed to determine the prevalence of inherited hemoglobin disorders in Yaoundé and Douala, Cameroon, as described elsewherehttp://emabs.lshtm.ac.uk/)45. This cohort was part of the VaccGene study as described elsewhere46. Blood samples were collected in Vacutainer tubes containing EDTA at birth and at subsequent birthdays up to 5 years of age. Anthropometry and biomarkers of iron and inflammation were measured in samples from a single annual visit based on the availability of stored samples. Serum ferritin , CRP , hepcidin (DRG Hepcidin-25 (bioactive) High Sensitive ELISA kit ), hemoglobin  and malaria parasitemia (Giemsa-stained thick and thin blood films) were measured. Genotyping of sickle cell trait in the VaccGene study is described below.EMaBS is a prospective birth cohort that was originally designed as an RCT to test whether anthelminthic treatment during pregnancy and early childhood was associated with differential response to vaccination or incidence of infections, such as pneumonia, diarrhea or malaria . Mothers of the infants were approached if the infants had received all of their EPI vaccines up to 6 months of age. The infants were sampled prospectively at 12 months after receipt of the measles vaccine at 9 months of age. Single whole-blood samples were collected in vacutainer tubes containing EDTA for measurement of iron and inflammatory markers and DNA extraction. Serum ferritin , CRP  and hepcidin (DRG Hepcidin-25 (bioactive) High Sensitive ELISA kit ) levels were measured. This cohort was part of the VaccGene study, and genotyping is described below.Infants born in Chris Hani Baragwanath Hospital living in the Soweto region of Johannesburg, South Africa were recruited from vaccine trials48. This study was designed to evaluate risks of cardiovascular disease as described elsewhere49. Serum ferritin (Roche immunoturbidimetric assay), CRP (ELISA) and hemoglobin  levels were measured from blood samples collected at a single clinic visit. Whole blood was used to extract DNA using Puregene reagents (Gentra Systems). Genetic studies were conducted as described elsewhere49, and rs334 genotypes were extracted from exome sequencing datasets as described in Peloso et al.50.This is a population-based longitudinal study of African Americans aged ≥21 years living in the Jackson, Mississippi metropolitan area in the USA51 as part of studies investigating the relationship between sickle cell trait, malaria and blood pressure53. Nairobi is located at a high altitude , and there is no evidence of malaria transmission there54. Population-wide censuses are conducted four times a year within the study area51. Using census data, we selected all school children aged 10–16 years who had a continuous record of residence since birth and had therefore had minimal exposure to malaria. To increase our efficiency in recruiting participants with sickle cell trait, we limited our recruitment to those who identified themselves as genetically descended from ethnic groups whose ancestral residence was in regions endemic for malaria . The frequency of sickle cell trait is much higher in these ethnic groups55. We measured ferritin, CRP  and hemoglobin  levels from stored blood samples. Sickle cell trait was typed by PCR36 using DNA extracted with the Qiagen DNA Blood Mini kit (Qiagen).Children aged 10–16 years were recruited from the Nairobi Urban Health and Demographic Surveillance System56. Sixty-two samples were randomly selected. Hepcidin levels were measured using the Hepcidin-25 (human) EIA Bachem kit and harmonized to DRG hepcidin values43. Severe malaria was diagnosed as P. falciparum parasites in the blood film plus clinical features of severe malaria, including hemoglobin levels <50gl-1, a hematocrit level of <15%  or a Blantyre coma score of 3 .We measured hepcidin levels in children with severe malaria admitted to Kilifi County Hospitaln =648) and the Illumina Multi-Ethnic Global Array (n =197) performed by the genotyping core facilities at the Wellcome Trust Sanger Institute. Genomic DNA underwent whole-genome amplification and fragmentation before hybridization to locus-specific oligonucleotides bound to silica beads with a 3-μm diameter. Fragments were extended by single-base extension to interrogate the variant by incorporating a labeled nucleotide, enabling a two-color detection . Genotypes were called from intensities using two clustering algorithms  in GenomeStudio version 2.0.5 (Illumina) incorporating data from proprietary predetermined genotypes. Details of genotyping and quality control are described elsewhere46. The variant rs334 was retained in all datasets following stringent quality control processes46.Sickle cell trait (rs334) single-nucleotide polymorphisms (SNPs) were directly genotyped in the VaccGene populations  using the HumanOmni 2.5M-8 (‘octo’) BeadChip array version 1.1 (Illumina)  in children <5 years or<15µgl-1 in children ≦5 years15. Anemia was defined as hemoglobin levels<110gl-1 in children aged<5 years or hemoglobin levels<115gl-1 in children ≦5 years17. In the JHS and Nairobi,ID was defined as ferritin levels<30µgl-1. In the JHS, anemia was defined as hemoglobin levels<120gl-1 in women or<130gl-1 in men17. IDA was defined as the presence of ID and anemia17. Malaria parasitemia was defined as a blood slide positive for asexual P. falciparum parasites. Underweight was defined as weight-for-age z score< -2 using WHO Growth Standards57.ID was defined according to WHO recommendation as ferritin levels <12µgl59, we further defined ID after regression correction for the effect of inflammation on ferritin levels as proposed by the Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia project60. This approach predicts what the ferritin level would have been in the absence of inflammation or infection and then applies the corrected values to estimate the prevalence of ID. The regression-correction approach followed a three-step process. In the first step, internal reference values for inflammatory markers (CRP or ACT) were defined as the tenth percentile. CRP levels were measured in all studies except in The Gambia, where ACT levels were measured. In addition to CRP, AGP was measured in Malawi, Ghana, DRC and Cameroon; however, because CRP was positively correlated with AGP, and also for consistency, we only corrected for CRP in these studies. To avoid overcorrection for very low levels of inflammatory markers, only participants with CRP or ACT values above the tenth percentile had their ferritin values subtracted from observed values in 19. In the second step, univariable linear regression models were applied to each study, with ferritin as the dependent variable, to estimate regression coefficients for the crude association between inflammatory marker and ferritin (β).In the third step, the regression coefficients estimated in step 2 were used to calculate adjusted ferritin values using As ferritin is an acute-phase reactant and correlates positively with inflammatory markers-1 in children <5 years or <15µgl-1 in children aged ≥5 years.We then defined ID using the regression-corrected unlogged ferritin value  using the same thresholds that were applied to the uncorrected ferritin levels in the WHO recommendations  and estimate a causal effect of that exposure on the outcome . To perform Mendelian randomization analyses, valid instrumental variables are required. The instrumental variables must be associated with the exposure of interest, and the effect size can be obtained from association studies. We therefore performed a systematic review and meta-analysis of published studies in African populations to determine the overall effect of common genetic polymorphisms that are associated with uncomplicated malaria. These polymorphisms included those influencing sickle cell trait,α-thalassemia and G6PD. We performed the search in the PubMed database for papers published before 20 March 2020. For sickle cell trait, the search terms included  or ‘malaria/blood’ (MAJR) or ‘malaria/ genetics’  terms) or ‘Malaria’ (MeSH) or ‘Malaria, Falciparum’ (MeSH) or ‘Plasmodium falciparum’ (MeSH)) and (‘HbAS’ (title/abstract) or ‘sickle cell trait’ (title/abstract) or ‘sickle cell trait/genetics’ (MeSH terms) or ‘sickle cell trait/blood’ (MeSH terms) or ‘Hemoglobin, Sickle’ (MeSH) or ‘Sickle Cell Trait’ (MeSH) and ‘Africa’ (MeSH terms)), yielding 594 articles. For α-thalassemia, the search terms included  or ‘malaria/ blood’ (MAJR) or ‘malaria/genetics’ (MeSH terms) or ‘Malaria’ (MeSH) or ‘Malaria, Falciparum’ (MeSH) or ‘Plasmodium falciparum’ (MeSH)) and  or ‘alpha-thalassemia/genetics’ (MeSH terms) or ‘alpha-thalassemia/ blood’ (MeSH terms) or ‘α-thalassemia’ (MeSH)) and Africa’ (MeSH terms) and yielded 65 articles. The search terms for G6PD included  or ‘malaria/blood’ (MAJR) or ‘malaria/genetics’ (MeSH terms) or ‘Malaria’ (MeSH) or ‘Malaria, Falciparum’ (MeSH) or ‘Plasmodium falciparum’ (MeSH)) and (‘G6PD’ (title/abstract) or ‘glucose-6-phosphate-dehydrogenase’ (title/abstract) or ‘glucose-6-phosphate-dehydrogenase/genetics’ (MeSH terms) or ‘glucose-6-phosphate-dehydrogenase/blood’ (MeSH terms)) and ‘Africa’ (MeSH terms) and yielded 187 articles.We restricted our analysis to studies conducted in Africa, as our outcome of interest (ID) was measured in African populations. We also focused on studies reporting IRR, as children are repeatedly infected with malaria; and, therefore, an IRR provides a better estimate of the true malaria risk reduction attributable to genetic polymorphisms. Studies included in the meta-analysis are shown in t-tests to test for differences in means of loge-transformed hepcidin concentrations between children with and without malaria parasitemia or between those with severe malaria and those without parasitemia. When appropriate, we fitted adjusted logistic regression models to determine the effect of sickle cell trait on ID. The cohort-specific ORs were meta-analyzed assuming fixed effects because there was little evidence of heterogeneity between studies. We used meta-analysis to determine the overall effect size and also to identify potential heterogeneity that may have been introduced by different populations and laboratory facilities. A P value <0.05 was considered statistically significant. All P values reflect two-tailed tests. Individuals with sickle cell disease (HbSS) were not included in the analyses, as numbers were few and there is little evidence suggesting that HbSS protects against uncomplicated malaria.All statistical analyses were conducted using Stata 13.0 (StataCorp). All measurements were taken from distinct samples. We conducted both cohort-specific and pooled analyses. When appropriate, we computed percentages, geometric means and deciles. We used two-tailed Student’s http://cnsgenomics.com/shiny/mRnd/) Mendelian randomization power calculator to calculate sample size. The sample size of 7,453 had power above 80% given the observed OR of the outcome variable per s.d. of the exposure variable of 2.65, 2% variation of clinical malaria that is explained by sickle cell trait62, 27% prevalence of ID and a type I error rate of 0.05.We used the online  –ID effect divided by the HbAS (rs334) –malaria effect. The first estimate (SNP –outcome) for sickle cell trait  on ID was from the above-described community-based cohorts in which the overall log odds estimate was determined using the meta-analyzed cohort-specific estimates  and (2) life-long adolescent residents of Nairobi (n=611), where there is no evidence of malaria transmissionTo determine whether sickle cell trait influences ID independently of malaria, we conducted sensitivity or zero relevance point analyses in negative controls, that is, populations that were not exposed to malariaIndividual study site ethical approvals were obtained for the Kilifi, Kenya study ), the Entebbe, Uganda study  and the Uganda National Council for Science and Technology (MV625) and in the UK by the London School of Hygiene and Tropical Medicine (A340) and the Oxford Tropical Research (OTR)  Ethics Committees), the Banfora, Burkina Faso study (by Ministere de la Recherche Scientifique et de l’Innovation in Burkina Faso (2014-12-151) and the OTR Ethics Committees(4112)), the Soweto, South Africa study (by the University of Witwatersrand Human Research (M130714) and the OTR Ethics Committees (1042-13 and 42-14)) and the West Kiang, The Gambia study ). For the additional seven study sites, individual study data transfer agreements were signed with the responsible study and/or institution’s principal investigator and the KEMRI-Wellcome Trust Research Programme study principal investigator, S.H.A. These studies had ethical approval to share de-identified data for further secondary analyses presented in this study. Informed written consent was obtained from all children’s parents or guardians.Further information on research design is available in the Nature Research Reporting Summary linked to this articleSupplementary information


Background: Antimalarial antibody measurements are useful because they reflect historical and recent exposure to malaria. As such, they may provide additional information to assess ongoing transmission in low endemic or pre-elimination settings where cases are rare. In addition, the absence of antibody responses in certain individuals can indicate the cessation of transmission. Commercial malaria enzyme-linked immunosorbent assays (ELISA) detect antimalarial antibodies and are commonly used to screen blood donations for possible malaria infection. However, there is no standardized test to detect antimalarial antibodies for epidemiological use. Here we compared five commercially available ELISA kits  in search of a standardized tool for supporting claims of absence of malaria transmission. For comparison, a research-based (RB) ELISA protocol was performed alongside the commercial kits.Results: The commercial kits were first compared using serum samples from known malaria-unexposed individuals (n = 223) and Toxoplasma-infected individuals (n = 191) to assess specificity and cross-reactivity against non-malaria infections. In addition, 134 samples from ≥10-year-olds collected in a hyperendemic region in the Gambia in the early 1990s were used to assess sensitivity. Three out of five kits showed high sensitivity (90–92%), high specificity (98–99%), low cross-reactivity (0–3%) and were considered user-friendly . Two of these kits (Trinity Biotech and NovaTec) were taken forward for epidemiological evaluation and results were compared to those using the RB-ELISA. Samples from two pre-elimination settings  were tested. Serological results from both the Trinity Biotech kit and the RB-ELISA concurred with recent passively detected case counts in both settings. Results from the Trinity Biotech kit reflected a significant decrease in the number of reported cases in Bataan in the 1990s better than the RB-ELISA. Results from the NovaTec kit did not reflect transmission patterns in either setting.Conclusion: The Trinity Biotech commercial ELISA kit was considered reliable for epidemiological use and accurately described transmission patterns in two (previously) malaria endemic settings. The use of this simple and standardized serological tool may aid national control and elimination programs by confirming that regions are free from malaria. A unique property of using antimalarial antibody responses as a measure of transmission is that when combined with age, they reflect the infection history of a given population –3. Antib19)  , 8 or sc19)  using an19) . The ELI19) . However19) , 13.There are several commercially available ELISA kits, for which production and operating procedures are standardized. Until now, these kits have been used to screen blood donations for evidence of malaria exposure prior to transfusion –20. In tToxoplasma-infected, malaria unexposed UK donors (to assess cross-reactivity). Malaria naivety was defined using a questionnaire to exclude malaria risk at the time of blood donation . To assess assay sensitivity, 134 samples collected from a hyperendemic region in the Gambia in the early 1990s were used. Sera were stored at −40°C until transportation on dry ice to the London School of Hygiene and Tropical Medicine (LSHTM) and stored at −80°C. Individuals were included only if they were 10 years or older by which exposure to malaria almost certainly would have occurred  as well as 191 samples from donation . ToxoplagM tests  and was occurred , 25. FurTwo study sites with contrasting malaria exposure histories and expected population immunity were selected. Firstly, samples were collected in Bataan, the Philippines in February 2017 . In this19 antigen, this sample size was expected to generate a 95% confidence interval for seroprevalence between 4.7% and 6.8% and for the seroconversion rate  between 0.0029 and 0.0043 for African settings, and 6.1 and 8.5% and 0.0029–0.0043 for non-African settings . Serum was separated at collaborating institutions in-country and were stored at −20°C until shipment on dry ice to the London School of Hygiene and Tropical Medicine, London, UK. Serum was stored at −20°C until sample processing.corr) according to the instruction manuals.Five commercial ELISA kits  were useP. falciparum antigens was used: apical membrane antigen 1 (AMA-1) (19) (corr) and normalized using the standard curve as previously described (A previously described research-based (RB) ELISA protocol , 3, 23 w (AMA-1) , the 19 -1) (19) , the ful-1) (19) , the ful-1) (19)  and glut-1) (19) . The coaAll statistical analyses were performed in R version 3.4.1 . Antibod2 tests for two-way contingency tables were used to compare seroprevalence among individuals who reported a history of malaria and those who did not; Fisher Exact Tests were used when the counts were below 5. Previous reports on malaria outbreaks in Cape Verde have identified malaria risk to be highest in adult men: more than two-thirds of the passively detected cases between 2007 and 2016 were in men older than 20 years  (n = 563) and women (n = 860) separately. A 5% significance level was used throughout the paper.The following serological metrics were used to show the presence of low-level recent transmission in Cape Verde and the absence of recent transmission in the Philippines: seroprevalence in children aged 1–5 years, SCRs and, where applicable, the presence of a change point in transmission. In addition, 95% Pearson-Clopper (exact) confidence intervals (CI) were used to quantify the uncertainty associated with seroprevalence. Pearson's χed risk) , 30. TheToxoplasma was high across most kits (>96%) except for the Cellabs kit . Receiver operating characteristic (ROC) curves for sensitivity and specificity estimates are included in Sensitivity, testing sera from individuals in a hyperendemic region in the Gambia in the 1990s as true positives, was high (90–95%) except for the DiaPro kit (86%); n = 4; 95% CI: 0.4–4.3%) for the Trinity Biotech kit; 4.7%  for the NovaTec kit; and 1.7%  for the RB-ELISA. The best fit seroconversion curve for the Trinity Biotech kit showed a change point at 22.0 years , though estimated at 41.5 years ago with a wider 95% confidence interval (28.5–45.5). The SCR estimates ranged from 0.001 annual seroconversion events per person (95% CI: 0.001–0.002) for the Trinity Biotech kit; 0.003 (0.002–0.004) for the RB-ELISA; and 0.013 (0.010–0.016) for the NovaTec kit. For all assays, seroprevalence in those with self-reported history of malaria (n = 87) was higher compared to those without (p < 0.001) but this difference was greatest for the Trinity Biotech kit  and for the NovaTec kit this was twelve (age range: 1–18). Very few were seropositive in multiple assays  and the RB-ELISA . This difference was not statistically significant for the NovaTec kit  and the RB-ELISA , corr 0.372). For the NovaTec kit, the SCR was zero for those aged 3 or less but with a wide 95% CI , low ease-of-use (DiaPro), high required blood volume (DiaPro) or production being discontinued (newbio). Further evaluation of kits in epidemiological characterization with samples from the Philippines and Cape Verde found that the Trinity Biotech kit described historical and recent malaria transmission patterns most accurately.There is historical evidence that demonstrating the absence of specific antibody responses can contribute to verifying areas or populations as malaria-free , 6, but Firstly, we used serum samples from a hyperendemic region and those from malaria naive populations to assess sensitivity and specificity. Specificity estimates for the kits reported here and from previous studies were largely similar to those reported in instruction manuals , 3. SpecOur rationale for extending technical performance evaluation with ease-of-use and costs was that whilst ELISA kits used for blood screening may have undergone some level of standardization, their intended use case scenario is different from epidemiological screening. Kits are typically designed to test batches of dozens to hundreds of samples, whereas a study to verify the absence of malaria transmission may include hundreds  or even P. falciparum alone, while the commercial kits additionally detect non-falciparum species (except for P. knowlesi). Historically, P. vivax was also present in Bataan like much of the Philippines.Although this was not an exhaustive assessment of all currently available commercial ELISA kits for antimalarial antibody detection, one of the five kits showed significant promise in correlating with described transmission patterns in two endemic settings. In Bataan, The Philippines, seroconversion curve profiles generated from the Trinity Biotech kit corresponded with a decrease in passively detected malaria cases in 1995 . SeropreFor Cape Verde, the low levels of transmission over the past decades were correctly identified as shown by the low, constant SCR recorded by the Trinity Biotech kit . The Trinity Biotech kit also identified a higher exposure to malaria in adult men  as previously described in epidemiological studies in Cape Verde , 30. SerThe dynamic range of OD values recorded in endemic populations was greatest for the Trinity Biotech kit and despite the use of neat serum, little to no background responses were seen , thus blBox 1Technological refinements- Broaden assessments of sensitivity and specificity from non-malaria infections- Optimize assays for use with dried blood spots- Evaluate options for scenario-specific assays by selecting antibodies with known exposure profiles  or specific Ig class responsesPlasmodium falciparum species- Confirm suitability for non-- Evaluate best methodology to determine seropositivity at (very) low transmissionProgrammatic questions- Revisit costs per sample (aiming for <1 USD/sample)- Establish limits of non-specific reaction - Establish sampling frames for specific epidemiological scenarios and disease transmission patterns; i.e., determine who and where to sample to confirm absence of malaria transmission - Improve understanding of relationship between seroprevalence, SCR and annual parasite incidence (API)/entomological inoculation rate (EIR) at (very) low transmissionP. falciparum, P. vivax, P. malariae, and P. ovale, but the accuracy of these assays in detecting exposure to P. knowlesi is unknown. The performance of the kits using DBS samples remains to be evaluated. Future work should focus on these technological refinements as well as outstanding programmatic questions relevant to the use of serological tools for certification of malaria-free populations.The Trinity Biotech commercial ELISA kit was considered most applicable for large-scale use in epidemiological surveys and accurately described malaria transmission in two pre-elimination settings. All the commercial ELISA kits studied reported the detection of four human malaria species: The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the LSHTM Research Ethics Committee (11684) for testing of anonymised UK donor samples collected by Public Health England/NHS Blood and Transplant. For the surveys in Cape Verde and the Philippines, ethical approval was obtained through the LSHTM Research Ethics Committee (11684), the Ethics Committee for Research in Health in Cape Verde (65/2016) and the Research Institute for Tropical Medicine in the Philippines (2016-26). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.CD, GS, NS, KT, AK, PC, and LH conceived the study. CD, GS, NS, KF, and FE designed the surveys. PB, JA, RR, MM, KF, JL, JF, JR, LG, NS, and GS performed field data collection. KT and SS provided antigen constructs. LH performed laboratory data collection with support from KT and TH. LH and CD analyzed and interpreted the data. LH wrote the first draft of the manuscript with support from CD. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer DN declared a shared affiliation, though no other collaboration, with KT and CD to the handling Editor.