Plasmodium falciparum. Among the five members of this family identified so far, only P. falciparum reticulocyte–binding homologue-5 (PfRH5) has been found to be essential for parasite survival across strains that differ in virulence and route of host-cell invasion. Based on its essential role in invasion and early evidence of sequence conservation, PfRH5 has been prioritized for development as a vaccine candidate. However, little is known about the extent of genetic variability of RH5 in the field and the potential impact of such diversity on clinical outcomes or on vaccine evasion. Samples collected during a prospective cohort study of malaria incidence conducted in Kalifabougou, in southwestern Mali, were used to estimate genetic diversity, measure haplotype prevalence, and assess the within-host dynamics of PfRH5 variants over time and in relation to clinical malaria. A total of 10 nonsynonymous polymorphic sites were identified in the Pfrh5 gene, resulting in 13 haplotypes encoding unique protein variants. Four of these variants have not been previously observed. Plasmodium falciparum reticulocyte–binding homologue-5 had low amino acid haplotype (h = 0.58) and nucleotide (π = 0.00061) diversity. By contrast to other leading blood-stage malaria vaccine candidate antigens, amino acid differences were not associated with changes in the risk of febrile malaria in consecutive infections. Conserved B- and T-cell epitopes were identified. These results support the prioritization of PfRH5 for possible inclusion in a broadly cross-protective vaccine.Reticulocyte-binding homologues (RH) are a ligand family that mediates merozoite invasion of erythrocytes in  Plasmodium falciparum, the deadliest human malaria parasite, has been limited in part by genetic diversity in the vaccine antigens in endemic areas,2 resulting in higher protection against parasites carrying alleles identical to the vaccine genotype.6 Molecular epidemiological surveys of P. falciparum genetic diversity at vaccine-testing sites can identify the most prevalent genetic variants of candidate antigens for consideration in designing broadly protective vaccines and assess the role of specific polymorphisms in strain-specific immunity.4The efficacy of vaccines targeting Plasmodium falciparum reticulocyte–binding protein homologue-5 (PfRH5) is a promising new blood-stage vaccine antigen that is moving quickly toward clinical development.9 The invasion of merozoites into the erythrocyte is a multistep process that starts with merozoite binding and reorientation onto the red blood cell (RBC), followed by the formation of a tight junction and culminates with parasite entry into the erythrocyte. Essential to this process is PfRH5, a 63-kDa protein encoded by a gene located in the subtelomeric region of chromosome 4. The gene (PF3D7_0424100) is 1,581 nucleotides long and is refractory to knockout in diverse strains.10 The erythrocyte receptor for RH5 is basigin (CD147), and antibodies specific to either RH5 or basigin inhibit erythrocytic invasion by P. falciparum in a dose-dependent manner in in vitro tests.11 Furthermore, IgG antibodies induced after PfRH5 immunization have higher potency for controlling RBC invasion by both homologous and heterologous P. falciparum strains in growth inhibition assays, as compared with IgG raised against the apical membrane antigen-1 (AMA1) and merozoite protein-1, two other leading blood-stage vaccine antigens.12pfrh5 gene exhibits limited polymorphism across multiple P. falciparum laboratory strains13 and geographic isolates.14 Moreover, allelic exchange experiments have determined that specific PfRh5 residues are critical for host erythrocyte invasion. For example, a nonsynonymous change in codon 204 (I204K) converted a poorly invasive strain into a highly invasive one.10 In a separate set of experiments, this invasion-enabling single nucleotide polymorphism (SNP) at position 204 changed the protein structure and greatly modified its binding specificity and infectivity.15 Taken together, these findings suggest that the functional requirement for erythrocyte invasion exert strong purifying selection on the locus and might constrain the genetic diversity of pfrh5. To evaluate the potential clinical implications of this hypothesis and to generate more evidence on the extent of natural diversity in PfRH5 at a single vaccine-testing field site, we investigated the prevalence and dynamics of pfrh5 polymorphisms during acute malaria episodes in Malian children and adults. Longitudinal analyses were carried out to assess whether specific pfrh5 alleles, or changes at this locus in consecutive infections, are associated with the development of malaria symptoms. This prospective study aimed to identify specific PfRH5 amino acid residues that are critical in clinical malaria, and to identify conserved B- and T-cell epitopes that should be taken into account in the design of a malaria vaccine intended to provide broader, strain-transcending protection against diverse parasites than was achieved with first-generation blood-stage malaria vaccines.Importantly, the 16 Briefly, 695 healthy children and adults, aged 3 months to 25 years, were enrolled in this ongoing study beginning in May 2011. Samples and clinical data for this study were collected from May 2011 through January 2012. Study participants were surveyed during scheduled visits occurring every 2 weeks and anytime they presented with malaria-like symptoms. During surveillance, clinical parameters were measured and malaria parasites were detected by thick smears. For this study, a clinical malaria episode was defined as any parasitemia by blood smear with a temperature greater or equal to 37.5°C. Participants with positive blood smears were treated according to the National Malaria Control Program guidelines in Mali. Peripheral whole-blood samples were collected by finger stick in capillary collection tubes  for the cryopreservation of the cell pellet and/or as dried blood spots (DBS) on 903 filter paper . Longitudinal polymerase chain reaction (PCR) analysis of DBS collected at scheduled visits was performed as previously described.17 Whole-blood samples from participants with at least two malaria episodes during the season were selected for genomic DNA (gDNA) extraction as described below  and hyperendemic, with seasonal peaks following the rains in July through October. The study population and clinical parameters have been described previously.ed below .pfrh5, a nested-PCR was used to amplify the major exon in all the samples. Briefly, primers F1 (5′ GCAATAAAAAAAACGAAGAATCAAGA 3′) and R1 (5′ TGAAAATATTCCATTTTAATTGGGAC 3′) were used in the primary PCR reaction . Conditions for this amplification were 94°C for 5 minutes, followed by 35 cycles of denaturation at 94°C for 1 minute; primer annealing at 48°C for 1 minute, and extension at 60°C for 1.5 minutes with a final extension at 60°C for 5 minutes. PCR products from the first amplification were subsequently used as a template for a second round of amplification  with primers F2 (5′ GAAAATAATCTGACGTTACTACCA 3′) and R2 (5′ GACATCATTGAACTTCATTTGTAG 3′). Amplification conditions were the same as in the primary amplification with only 10 cycles instead of 35. PCR reactions were carried out in a total volume of 50 μL with 47 μL of Platinum Super Mix in 96-well plates , 1 μL of each primer  and 1 μL of malaria parasite DNA. Secondary PCR had 47 μL of Platinum Mix, 1 μL of each  primer and 1 μL of primary PCR product. Secondary PCR products were electrophoresed on a high-throughput precast gel system (Invitrogen), then visualized and photographed under ultraviolet light with ethidium bromide as the stain.Whole-blood gDNA was extracted from blood samples and DBSs using the QIAmp 96 DNA kit . To genotype polymorphisms in P. falciparum 3D7 reference (GenBank accession number: XM_001351508) sequence using Sequencher v.5.0 . Infections were classified as single/predominant allele infections or as multiple allele infections if the secondary peak height was greater than 50% of the primary peak height at any polymorphic position. Sequence alignment was performed using Bioedit version 7.18PCR products were purified using filter plates  attached to a vacuum manifold and eluted in water, and sequenced in a 10 μL reaction using BigDye v3.1 . The sequencing products were ethanol precipitated and run in 7 μL HiDi formamide on an ABI 3730XL 96-capillary sequencer . Following sequencing, fragments were assembled, edited and aligned to pfrh5 locus of the P. falciparum 3D7 strain (GenBank accession number: XM_001351508) was used as the reference sequence for all the comparisons. The nucleotide diversity at the locus, among sequences generated from samples collected during the study, was assessed by estimating the parameter π, the average number of nucleotide differences per site between two random sequences.19 Bootstrap resampling was used to simulate the distribution of π and to calculate confidence intervals (CIs). These analyses were conducted using MEGA620 and DnaSP v5.21 The prevalence of nonsynonymous substitutions was estimated using SAS v9.1 . To assess if regions of Pfrh5 evolve under neutrality (Kimura 1983), the McDonald and Kreitman test was measured using Plasmodium reichenowi (PRCDC_0322700) as an outgroup, and analyses were performed using DnaSP v5.21 The hypothesis that positive selection has occurred in PfRH5 branch/branches tree was assessed by applying the branch-site test22 and the branch-site unrestricted statistical test for episodic diversification (BUSTED) implemented in the HYPHY package.23 Although BUSTED identifies the effect of sporadic positive selection on a subset of branches in the lineage, the branch-site test uses a χ2 distribution with one degree of feedom to compare a model, which allows positive selection on one or more variants of a group to a model that does not permit positive selection (P < 0.05 for sites positively selected). Tajima’s D statistic was also calculated, with a positive value of Tajima’s D indicating an excess of alleles at intermediate frequency, consistent with balancing selection, and negative values consistent with directional selection or with rapid population expansion following a bottleneck. To assess recombination events in pfrh5, which may result in negative Tajima’s D values and lead to a false inference of directional selection or rapid expansion, an estimate of recombination (R) and the minimum number of recombination events (RM)24 in the data were estimated using DnaSP v5. These recombination events were confirmed by the genetic algorithm recombination detection test.25 Genetic association between polymorphic sites and the effect of intragenic recombination on sequence polymorphism were assessed using the Zns27 statistics, respectively.Haplotypes were defined as an uninterrupted sequence of nucleotides or amino acid sequence variants, and the 28 which treats the number of populations as a random variable, was used to determine the posterior number of populations (K). We identified two populations by this Dirichlet process. We then used Structure version 2.3.329 to assign Rh5 sequences to haplotype groups. Ten runs of 50,000 burn-ins and 100,000 iterations were performed for K = 2 using an admixture model.To group PfRH5 sequences, a sequence clustering analysis was conducted. Structurama, a Bayesian clustering algorithm software,N = 13), and of amino acid residue polymorphic sites (N = 10) were compared in clinical and asymptomatic malaria cases using a χ2 test while correcting for multiple measurements . Generalized estimating equations were used to perform logistic regression to estimate whether specific pfrh5 alleles or shifts in pfrh5 alleles were associated with the development of clinical symptoms in an individual’s consecutive infections. Both single- and multiple-clone infections were included in the analysis. The primary outcome was whether an individual’s second of two paired consecutive infections was symptomatic or asymptomatic, with the presence of infection being defined based on the results of the pfrh5 PCR (N = 40); absence of infection to symptomatic ; asymptomatic to asymptomatic ; asymptomatic to symptomatic ; symptomatic to asymptomatic ; and symptomatic to symptomatic . The primary predictor for the analysis was the presence of a specific pfrh5 variants or whether there was a change in amino acid sequence variant in the next consecutive infection. The models included potential confounding variables such as age group, or symptoms in the first of the two infections. Analysis was performed using SAS v9.1.The frequency of protein sequence variants, or haplotypes  using a threshold of 0.85. A T-cell epitope prediction algorithm  was used to screen PfRH5 sequences for potential CD4+/CD8+ epitopes with respect to the most frequent human leukocyte antigen (HLA) allele frequencies in Mali.31 Disordered regions  were identified using the regional order neural network (RONN) server (http://www.strubi.ox.ac.uk/RONN). A putative epitope cutoff was set at 50 nM (high affinity binding).The study protocols were reviewed and approved by the institutional review boards of the University of Sciences, Techniques, and Technology of Bamako, Mali; the University of Maryland Baltimore; and the National Institute of Allergy and Infectious Diseases. The study participants gave their consent and/or assent to participate in the study.N = 312) were selected from the incidence study sample repository. Samples had been collected biweekly from individuals with at least two malaria episodes during study follow-up starting at the beginning of the 2011 malaria transmission season. Of these specimens, 198 (63.5%) were successfully sequenced using primers specific to the Pfrh5 gene. The dynamics of consecutive malaria infections and clinical episodes was assessd using 139 sequences generated from paired-consecutives episodes as described in Blood spots on filter paper and/or whole-blood specimens  followed by H148D (5%). Based on these 10 amino acids polymorphisms, 13 unique protein variants were identified . Interestingly, the incidence of the two major haplotypes fluctuate slightly and alternate over time .Ten nonsynonymous polymorphisms were identified in entified . Seven oentified . An analver time , which ipfrh5 gene. At this study site, pfrh5 nucleotide diversity (π ± SD) and haplotype diversity (h ± SD) were 0.00061 ± 0.00005 and 0.58 ± 0.027, respectively. All polymorphisms in the data occurred at nonsynonymous sites, with πS = 0 and πN = 0.00075. The average number of pairwise nucleotide differences between sequences in this sample set was 0.74.Genetic diversity parameters were estimated for the whole pfrh5 and influence its eventual use as a vaccine candidate, the McDonald and Kreitman test was conducted with P. reichenowi used as a source of the divergence estimates to be compared with polymorphism in P. falciparum. Despite the higher rate of nonsynonymous polymorphism relative to synonymous polymorphism, the test does not support a departure from neutral expectation . This may result from the lack of power due to the small number of polymorphic sites, which was exacerbated by the elimination from the test of three codons with multiple variable positions. Moreover, the estimate of the coefficient of variation for αs generated using a maximum likelihood model (gamma distribution with shape 1) was small (< 0.000001) inferring a constant evolution rate among sites. Finally, the Tajima’s D statistic did not show clear departure from neutrality . Despite not being significant, the strong negative Tajima’s D value may be suggestive of a rapid population expansion following a bottleneck.To assess whether positive selection might be responsible for the low diversity observed in pfrh5 diversity was estimated. Sequence analyses suggested that the overall recombination parameter (R), which is the recombination rate per generation between the most distant sites of Pfrh5, was 77.3. An estimate of R between adjacent sites of Pfrh5 was 0.064, whereas the minimum number of recombination events (Rm) in the history of the dataset was one. These findings are indicative of meiotic recombination occurring within the Pfrh5 locus. Values of the genetic association estimated between polymorphic sites (Zns) and the effect of intragenic recombination (ZZ) on sequence polymorphism were 0.0216 and 0.0256, respectively, indicative of low linkage disequilibrium between polymorphic sites.Recombination is a process that creates new haplotypes by reassorting variants of a locus. These novel haplotypes may be different from the vaccine allele(s), limiting its efficacy. To evaluate this second source of genetic diversity in the dataset, the contribution of recombination to pfrh5 alleles were associated with the development of clinical symptoms in consecutive infections. The primary predictor for the analysis was the presence of a specific pfrh5 allele. Only amino acid 203 had enough changes during follow-up to assess the dynamics of codons in relation to the occurrence of malaria clinical episode. Results from the regression models for amino acid 203 (95% CI: −1 to 1.1) analysis did not show an association between amino acid residue at position 203 and the occurrence of clinical malaria episode in consecutive P. falciparum infections. Finally, a comparison of mean parasite density in mutated  and wild type  parasites at position 203 revealed that neither of the amino acids at this site was associated with the level of parasitemia.Participants who had clinical malaria experienced one to five malaria episodes during the 9-month study period. Nearly half (48%) of these participants experienced three malaria episodes. A logistic regression model was used to estimate whether specific 2 = 2.46, P = 0.12) nor variant B  was associated with malaria risk. Furthermore, the incidences of two of the most prevalent amino acid polymorphisms, H148D and C203Y, were identical  in study participants when malaria clinical cases were compared with asymptomatic infections .To investigate whether individual codons or PfRH5 variants affected the risk of clinical malaria episodes, association between the most prevalent PfRH5 variants and residues and the incidence of clinical malaria was evaluated. Neither variant A   class I-restricted T-cell epitopes, 175 MHC class II-restricted T-cell epitopes, and three IURs (highly disordered) were identified , Table 3= 0.004) , revealiPlasmodium falciparum reticulocyte–binding homologue-5 is a conserved malaria vaccine candidate that induces antibodies that are protective in in vitro assays,32 non-human primate studies7 and field studies.33Plasmodium falciparum reticulocyte–binding homologue-5 interacts with basigin (CD147), an erythrocyte surface protein, to allow the invasion of the malaria parasite into the erythrocyte. The goal of this study was to assess the genetic diversity, haplotype prevalence, and dynamics of the PfRH5-encoding gene to guide the selection of variants and/or B- and T-cell epitopes that are representative of the parasite population of this study site for possible inclusion in a broadly effective malaria vaccine. Examining the dynamics of antigen diversity in prospective cohort studies can show evidence to guide the design of vaccines that will be efficacious against diverse targets. Clinical trials have shown that some malaria vaccines based on a single strain induce protection that is specific to the vaccine strain.36 Likewise, pneumococcal37 and influenza38 vaccine trials have shown the importance of antigen polymorphisms on vaccine efficacy and the selection of variants to include in vaccine formulations.39 our large field study confirmed that pfrh5 was highly conserved in a setting where other vaccine antigens show extensive polymorphism. The nucleotide diversity, as measured by π, was very low (0.00061 ± 0.000012) compared with the diversity observed in the locus encoding the well-known blood-stage AMA1 (0.0163) from the same geographical region.40 Despite the high rate of polymorphism in nonsynonymous relative to synonymous sites, usually suggestive of positive selection, none of the statistical inference methods used to detect departure from neutrality was indicative of evolution under immune pressure, possibly because the tests were underpowered to detect it in this dataset. In addition, recombination events that may introduce new protein sequence variants in the locus were very low (only one event observed), as indicated by low R, Rm, Zns, and ZZ statistics. The low effective recombination rate may also be explained by the limited number of variable sites that are not singletons, which prevents the detection of recombination events. The very low values of nucleotide diversity not only in nonsynonymous (πNS) sites but also in silent sites (πS) and observed in this dataset are usually indicative of a very recent common ancestor to all sequences, as one might expect from a hard sweep or a recent, strong bottleneck. This observation is consistent with a previous study that suggests that a small chromosomal region containing Pfrh5 and three other genes was recently acquired via lateral gene transfer from another Plasmodium lineage, possibly an ancestor of Plasmodium adleri,41 an event that would impact polymorphism similarly to a very recent bottleneck.In the present study, we analyzed 198 sequences from Kalifabougou, Mali. Power calculations based on sequences extracted from MalariaGEN database show that, with this sample size, we have 83.6% power to identify 85.7% of all alleles present, suggesting that this sample size provides a reliable assessment of population diversity. Two protein variants that represent most of the circulating alleles in this region were identified. In support of previous findings based on small numbers of parasite strains,pfrh5, which gave rise to 13 protein variants with frequencies ranging from 0.5% to 57%. In comparison, using sequences from the blood-stage vaccine candidate AMA1, 186 unique amino acid haplotypes were identified in Bancoumana 40 and 214 unique haplotypes were observed in Bandiagara ,5 two other towns in Mali. Plasmodium falciparum reticulocyte–binding homologue-5 amino acid polymorphism C203Y, located near the boundary of the region involved in the PfRH5–basigin interaction was the most variable residue observed in this dataset. None of the codons within PfRH5-basigin interface 42 were polymorphic. The conservation of these residues is suggestive of the functionally critical role they play in the interaction between PfRH5 and basigin to allow parasite invasion into RBCs. The overall conservation of pfrh5 is highly unusual for what would be expected of a gene with high expression during the blood-stage and for which the product is exposed to the host’s immune system, and therefore more inclined to exhibit a high degree of amino acid diversity.44 However, as mentioned previously, these observations may be more reflective of the unusual evolutionary history of the pfrh5 locus in P. falciparum41 than of its interactions with the human immune system.Ten nonsynonymous SNPs were identified in P. falciparum reticulocyte–binding protein homologue-5-interacting protein45 and P113.46 Therefore, polymorphisms in this protein may be found at flexible or exposed sites. Findings from Kenya47 and Mali,16 suggest that anti-PfRH5 antibody concentrations were very low in malaria-exposed subjects compared with other blood-stage antigens, providing support for the notion that PfRH5 is either non-immunogenic or that its immunogenicity is undermined by mechanisms that reduce immune recognition or response. The low antigenicity of PfRH5 in natural settings will likely require the use of a potent adjuvant in a RH5-based vaccine to induce the high antibody levels needed for protection.It is also possible that the protein has limited exposure to the immune system as it has to form a complex with pfrh5 alleles were associated with clinical disease, suggesting that certain alleles may allow for more efficient erythrocyte invasion and potentially more virulent infections. This study did not identify amino acids that were associated with the parasite’s ability to invade RBCs, at least as measured by association with clinical outcomes. All residues at position 204 in this dataset were shown to be wild type, suggesting a high relative fitness of an isoleucine (I) in this position. Although this post hoc analysis was based on a relatively small number of paired consecutive infections, the very low degree of polymorphism in PfRH5 compared with other blood-stage antigens supports the conclusion that not enough time has occurred for genetic variants to emerge.Using naturally-occurring clinical malaria episodes, we assessed whether specific Identifying epitopes that are conserved and localized in intrinsically unstructured regions of PfRH5 has important implications for vaccine design. Our study revealed 21 B- and T-cell epitopes that are conserved and located in disordered regions across all the sequences from the Kalifabougou site. Based on our data, the inhibition activity of these epitopes and/or fragments should be assessed by functional assays to ascertain their relevance as candidate epitopes of a PfRH5-based vaccine.48 The results of this study suggest that polymorphism in PfRH5 is very limited, stable during consecutive infection that occur over the course of a malaria transmission season and not associated with clinical risk. This high degree of conservation and lack of association between genetic variation and clinical disease is in sharp contrast to other leading blood-stage vaccine antigens whose efficacy was constrained by allelic escape.50 Although encouraging for PfRH5’s prospects as a vaccine, even this limited degree of polymorphism allows for the possibility of vaccine escape and allele-specific efficacy under the intense selection of vaccination as supported by the apparent signal of frequency-dependent selection. Moreover, although changes in the P. falciparum circumsporozoite protein (CSP) were not associated with clinical risk in paired consecutive infections,51 the CSP-based vaccine RTS,S nevertheless did have allele-specific efficacy.6 If the predominant alleles identified in this study are confirmed to predominate in other epidemiological settings, it would be prudent to include both alleles in a vaccine containing PfRH5 to reduce the effect of allele-specific efficacy.We have advocated routinely assessing whether polymorphism in vaccine candidates is immunologically important and therefore relevant to vaccine development. The data reported here on the diversity and dynamics of this antigen’s alleles and their association with the hazard of clinical malaria episodes may inform the design of a PfRH5-based vaccine or, more likely, of a multivalent, multistage malaria vaccine that improves on the modest efficacy of the first-generation vaccines.


Plasmodium falciparum infection. Genome-wide linkage studies have mapped mild malaria resistance genes on chromosome 6p21, whereas NCR3-412 polymorphism (rs2736191) lying within this region was found to be associated with mild malaria.Host factors, including host genetic variation, have been shown to influence the outcome of Plasmodium falciparum malaria patients . NCR3-412 (rs2736191) was analysed by sequencing, and haematological parameters were measured. Finally, their association with clinical phenotypes was assessed.Blood samples were taken from 188 NCR3-412 with either cerebral malaria, severe non-cerebral malaria, or severe malaria after grouping cerebral malaria and severe non-cerebral malaria patients.We evidenced an association of thrombocytopenia with both cerebral malaria and severe non-cerebral malaria, and of an association of high leukocyte count with cerebral malaria. Additionally, we found no association of NCR3 genetic variation has no effect, or only a small effect on the occurrence of severe malaria, although it has been strongly associated with mild malaria. We discuss the biological meaning of these results. Besides, we confirmed the association of thrombocytopenia and high leukocyte count with severe malaria phenotypes.Our results suggest that  Plasmodium falciparum malaria remains a major public health concern causing 212 million malaria cases occurred in 2015  alters both the binding of transcription factors to the DNA and the promoter activity. Moreover, NCR3 is involved in the recognition of P. falciparum- infected red blood cells by natural killer (NK) cells. It might be possible that NCR3-412 alters the transcriptional level of NCR3 resulting in an effect on the amount of NKp30 at the NK cell surface and their response to infected red blood cells. To our knowledge, there was, however, no NCR3 association study with severe malaria.ina Faso  and in tina Faso . Both poNCR3 as a candidate gene for severe malaria, and analysed its polymorphism in P. falciparum-infected individuals. We genotyped NCR3-412 (rs2736191) in DNA samples from patients including 76 mild malaria, 85 cerebral malaria, and 27 severe non-cerebral malaria subjects. We report the analysis of NCR3-412 (rs2736191) and several haematological parameters association with severe malaria phenotypes.Here we considered P. falciparum infection was determined by an immunoassay enabling the detection of pfHRP2 . P. falciparum parasitemia was assessed by microscopic examination of thin and thick smears, prior anti-malarial treatment. Clinical histories, data and basic demographic information, ethnic group, and study questionnaires were recorded at baseline for each subject. In particular, the affiliation to an ethnic group was provided by the patients or their parents. At the day of admission, biological data including parasite density, haematology and biochemistry parameters were determined at the hospitals’ medical laboratories and recorded. Briefly, thick and thin blood films were stained with 10% Giemsa solution for 15 min to measure parasitaemia. Parasite determination and measurement were established by two independent readings. The parasitaemia was defined as the number of parasitized erythrocytes observed per µl of blood. P. falciparum was the only Plasmodium species detected. Samples were taken at the admission before any treatment. An informed consent was obtained from each participant and/or their relatives prior to inclusion, after giving them written or verbal information in their native language. The protocols were approved by the investigator’s institutions, the National Ethical Committee and the Ministry of Health of Senegal.The study was performed between 2012 and 2015 in two Senegalese hospitals including one in Dakar and the other in Tambacounda at the Southeast part of the country. Two categories of patients with clinical malaria namely, severe malaria (SM) and mild malaria (MM) patients were enrolled. All the clinical cases were defined according to the WHO criteria . For allP. falciparum infection, without other clinically cause of impaired consciousness, such as hypoglycemia, meningitis, and encephalitis according to WHO criteria  and non CM (NCM) cases. CM cases were defined on the basis of a deep coma, an unpurposeful response or no response to a painful stimulus by Glasgow score <9, a microscopically diagnosed criteria . NCM casP. falciparum parasitaemia of <25,000 parasites/µL of blood, with no evidence of impaired consciousness or seizures before and at the time of enrolment. MM patients were followed up for two weeks after their enrolment, and there was no case of impaired consciousness or seizures. Blood samples from MM patients were obtained on the day of hospital admission.Regarding MM, a total of 76 patients who were treated at the outpatient clinic of the hospital were initially enrolled. Of these, 76 patients had fever with Blood Mini Kit  was used for DNA extraction according to the manufacturer’s instructions . The totNCR3-412 (rs2736191) was designed as described (rs33930165), the forward primer was 5′-CTGAGGGTTTGAAGTCCAAC- 3′, and the reverse one was 5′-CAGCATCAGGAGTGGACAG-3′. The PCR products were submitted to electrophoresis on 1.5% agarose gel electrophoresis to verify the product size and were purified using a PCR DNA Purification Kit (QIAGEN). The purified PCR products were sequenced using the Sanger method .The primer pair to amplify DNA fragments containing escribed ; PCR amprs2736191 polymorphism with malaria phenotypes, we conducted a chi-square test and a logistic regression analysis, and we evaluated the odds ratios (OR) and their 95% confidence intervals (CIs). MM group was considered the control group, whereas the case group consisted of CM patients, NCM patients, or severe malaria patients after grouping CM and NCM patients. The distribution of MM patient genotypes was compared with the one of CM, NCM, or severe malaria patient genotypes. All the tests were two-sided. G*power software was used to compute statistical power for the genetic association study.Hardy-Weinberg equilibrium was tested and allelic frequencies were calculated, as described . Other sdf = 2, P = 0.3). CM patients had a significantly higher risk of dying compared to NCM (P = 0.04) or MM patients (P = 2.0 10−6) by using a Fisher exact test. Moreover, there were significant differences between CM, NCM, and MM patients on the basis of the Kruskal-Wallis test for age (P = 0.009), erythrocyte count (P = 0.019), haematocrit (P = 3.3 10−6), haemoglobin concentration (P = 2.4 10−6), leucocyte count (P = 0.006), and platelet count (P = 7.9 10−4).P < 0.006) by using the Mann–Whitney method, whereas there was no difference between CM and MM for age. Erythrocyte count (P = 0.01), haematocrit (P = 4.9 10−6), and haemoglobin concentration (P = 1.4 10−6) of NCM patients were lower than those of MM patients. NCM patients showed a lower haematocrit (P = 1.1 10−4) and a lower haemoglobin concentration (P = 3.4 10−5) compared to CM patients. Moreover, we detected a reduction of platelet count (P = 0.0003) and an increase of leucocyte count (P = 0.0015) in CM patients compared to the ones of MM patients, as shown in P = 0.14) and for platelet count (P = 0.64). Furthermore, we found a reduction of platelet count in NCM patients compared to the ones of MM patients (P = 0.018), whereas there was no difference for the leucocyte count (P = 0.44) between these two groups.NCM patients were younger than CM and MM patients , whereas there was a trend when comparing Peul to all other ethnic groups . More specifically, there was a trend in favour of a better protection from CM , although not significant (P > 0.05), on the basis of the comparison of MM with CM patients. In the same way, there was a trend in favour of a lower parasitaemia mean in Peul patients, although not significant (P > 0.05); there was, nevertheless, a lower parasitaemia variance in Peul patients (P = 0.003). Haemoglobin S and haemoglobin C carriers were rare in CM, NCM, and MM patients, as shown in NCR3 genotype by clinical status. There were 123 GG, 52 GC, and 8 CC individuals. NCR3-412 was in Hardy-Weinberg equilibrium (P = 0.77). NCR3-412G allele frequency was 0.81, whereas NCR3-412C allele frequency was 0.19. We clustered GC and CC individuals for further analyses.NCR3-412C allele carriage with severe malaria by using the Chi-square test. To this aim, we compared the distribution of MM patient genotypes with the one of severe malaria patient genotypes. There was no association with CM , NCM , or severe malaria after grouping CM and NCM patients .We tested the association of the P = 0.56), NCM (P = 0.92), or severe malaria (P = 0.61). The odds of CM, NCM, and severe malaria between NCR3-412GG and NCR3-412GG/GC were 0.813 , 1.047 , and 0.847 , respectively. Similar results were obtained after adding haemoglobin genotypes and ethnic groups as covariates. In addition, we assessed the power of our analyses on the basis of the sample size used, an α-risk of 5%, and an effect size equal to the one detected by NCR3-412C allele carriage with mild malaria. We obtained a power of 85% and 90% for CM and severe malaria, respectively.We applied a logistic regression method when taking into account age and gender, which may influence malaria phenotypes. There was no association with CM (NCR3 genetic variant, which was linked and associated with mild malaria in Burkina Faso (NCR3-412, haemoglobin C and haemoglobin S polymorphism in patients with either mild or severe malaria, and performed an association study based on a case-control design.A few genes have been associated with both mild malaria and severe malaria. We investigated a ina Faso . We recrin vitro in human brain microvascular endothelial cells (Reduced red blood cell count, haematocrit, and haemoglobin concentration were associated with noncerebral malaria, indicating a marked anaemia in these patients compared to mild malaria and cerebral malaria. Thrompocytopenia was associated with both cerebral malaria and severe non-cerebral malaria, as previously reported . Thrompoal cells , thus coMoreover, an increase of leukocyte count was associated with CM, whereas MM patients and NCM patients did not differ in their leukocyte count. In the same way, leukocyte counts were reported to be higher in severe malaria patients compared to those of mild malaria patients , and in Since the Peul ethnic group which has a particular genetic background has a lower risk of mild malaria attacks, a lower parasitaemia, and a higher anti-plasmodial antibody response compared to other sympatric ethnic groups , we hypoSince, haemoglobin S and haemoglobin C are well known to protect against severe malaria , we searNCR3-412 with severe malaria. Mild malaria patients and severe malaria patients did not differ in their NCR3-412 allelic frequency. Power studies revealed a power of approximately 85% based on an effect size corresponding to the one found in the mild malaria studies (We further assessed the association of  studies . This inNCR3-412 and severe malaria, we should keep in mind that there is an association between NCR3-412 and mild malaria (NCR3-412 polymorphism may partly explain why individuals with asymptomatic malaria have mild malaria attacks, but is unlikely to explain why some patients with mild malaria develop severe forms of the disease (If we assume that there is no association between  malaria . This me disease .It is important to point out the limitation of our study. First, the time of our clinical investigation was limited. Also, the absence of CM or NCM was registered on the basis of the declaration of the patients or their parents. Second, it cannot be excluded that clinical status could be due to differential anti-malarial immunity response resulting from different exposure levels. Finally, the sample size of our study population is limited. In spite of this limitation, it should be stressed that we detected an association of leucocyte and platelet counts with clinical status, confirming previous results. This shows that the sample size was sufficient to allow us to detect association with a high effect size. In this way, our power analysis for genetic association indicated that we could detect an association of NCR3 with severe malaria when assuming an effect size similar to the one we calculated for mild malaria in Burkina Faso or in the Republic of Congo . NeverthNCR3 genetic variation. This suggests that genetic variation at this locus does not influence the molecular mechanisms involved in the most severe forms of the disease, although it influences the risk of developing mild malaria attacks.To summarize, we conducted an epidemiological study that allowed us to confirm the association of cerebral malaria with thrombocytopenia and leukocyte count. Besides, we did not detect an association of severe malaria with 10.7717/peerj.6048/supp-1Data S1Gender, age, ethnic group, clinical malaria phenotype, survival, NCR3 and HBB genotypes are stated for each individual.Click here for additional data file.


Plasmodium falciparum malaria is mainly mediated by IgG antibodies but the subclasses, epitope targets and effector functions have not been unequivocally defined. Dissecting the type and specificity of antibody responses mediating NAI is a key step toward developing more effective vaccines to control the disease. We investigated the role of IgG subclasses to malaria antigens in protection against disease and the factors that affect their levels, including vaccination with RTS,S/AS01E. We analyzed plasma and serum samples at baseline and 1 month after primary vaccination with RTS,S or comparator in African children and infants participating in a phase 3 trial in two sites of different malaria transmission intensity: Kintampo in Ghana and Manhiça in Mozambique. We used quantitative suspension array technology (qSAT) to measure IgG1−4 responses to 35 P. falciparum pre-erythrocytic and blood stage antigens. Our results show that the pattern of IgG response is predominantly IgG1 or IgG3, with lower levels of IgG2 and IgG4. Age, site and RTS,S vaccination significantly affected antibody subclass levels to different antigens and susceptibility to clinical malaria. Univariable and multivariable analysis showed associations with protection mainly for cytophilic IgG3 levels to selected antigens, followed by IgG1 levels and, unexpectedly, also with IgG4 levels, mainly to antigens that increased upon RTS,S vaccination such as MSP5 and MSP1 block 2, among others. In contrast, IgG2 was associated with malaria risk. Stratified analysis in RTS,S vaccinees pointed to novel associations of IgG4 responses with immunity mainly involving pre-erythrocytic antigens upon RTS,S vaccination. Multi-marker analysis revealed a significant contribution of IgG3 responses to malaria protection and IgG2 responses to malaria risk. We propose that the pattern of cytophilic and non-cytophilic IgG antibodies is antigen-dependent and more complex than initially thought, and that mechanisms of both types of subclasses could be involved in protection. Our data also suggests that RTS,S efficacy is significantly affected by NAI, and indicates that RTS,S vaccination significantly alters NAI.Naturally acquired immunity (NAI) to  Plasmodium falciparum is a significant health problem particularly in children under 5 years old in sub-Saharan Africa, with about 219 million cases and 435,000 deaths worldwide  to malaria is acquired with age and exposure  plus epitopes from the CSP carboxy-terminal, targeting the sporozoite, and liver stages of infection, and fused to the S surface antigen of hepatitis B (HBsAg) virus and coexpressed with HBsAg alone. In the phase 3 trial, RTS,S was formulated in the AS01E liposomal adjuvant containing monophosphoryl lipid A and QS21 and was designed to induce strong anti-CSP antibody and T helper 1 cell responses . In the context of the African pediatric multicenter RTS,S/AS01E phase 3 trial for licensure : Kintampo in Ghana  and Manhiça in Mozambique (representative of low MTI)   to be abn = 195). For the correlates of malaria protection and risk analysis, 78 children and infants were randomly selected from Kintampo, and 117 participants were selected from Manhiça according to a prior case-control study of cellular markers (Children 5-17 months and infants 6–12 weeks at recruitment with ≥150 μL plasma/serum samples available at M0 (baseline) and M3 were selected. We included 129 RTS,S/AS01E— and 66 comparator— vaccinated subjects from both sites  and glutathione S-transferase (GST) coupled beads for background determination and as a control for signal from non-specific binding of P. falciparum GST fusion proteins, respectively. P. falciparum proteins were covalently coupled directly to MagPlex beads and blocked with BSA. qSAT assays were previously standardized and optimized to control for sources of variability , by approach  consider10 MFI) as an outcome and including the following predictors: vaccination, sex, malaria transmission season at M3, having clinical malaria episodes between M0 and M3, and baseline variables like age (in weeks), antibody levels (log10 MFI), hemoglobin (Hb) concentration, weight-for-age Z score (WAZ), and height-for-age Z score (HAZ). Models were also fitted separately at pre-vaccination. Malaria transmission season was defined as high between April-October for Kintampo, and November-April for Manhiça; the remaining months were defined as low transmission. The effect of baseline antibody levels  was evaluated using the same antigen/Ig as the outcome variable at M3. The purpose of presenting the analysis of the antibodies at baseline (before vaccination) is that our prior studies showed that they have a very significant influence on the M3 antibody levels and subsequent risk of clinical malaria  using M3 antibody levels  were fitted first univariable and next multivariable to obtain the effect of different predictors in the odds of having malaria. Main predictors included levels (log10MFI) of antibodies at M3, and increment (log10-transformed) of antibody levels between M0 and M3. The impact of the other covariates (same as above) on the association between antibody responses and malaria risk/protection was also assessed. The linearity of the log10-transformed antibody levels was evaluated when the outcome was case-control.Analysis of antibody correlates of protection was based on a case-control design. The outcome was clinical malaria detected by PCD defined by fever >37.5°C with any parasitemia (without a detection limit) in the 12 months after the start of follow-up (M3 plus 14 days). Logistic regression models  and Partial Least Squares Discriminant Analysis (PLS-DA) using the R packages FactoMineR  and Discn = 29], 21% in Manhiça [n = 6]), and the remaining in the infant age cohort. Parasitemia of subjects who had clinical malaria was comparable between RTS,S and comparator vaccinees. The Kaplan Meier median follow-up time was 365 days (IQR = 128 to 365).RTS,S and comparator vaccinees were similar with regards to baseline characteristics  and most of them (93%) completed the 12-month post-vaccination follow-up . The medWe detected IgG responses to most malarial antigens with a predominance of the IgG1 subclass, followed by IgG3, although some antigens  had similar levels of IgG1 and IgG3 , MSP1 bl2 Mad20, EBA140 R3-5, and RH4.2.Comparing antibody profiles over time, IgG levels significantly decreased from M0 to M3 for some antigens probably reflecting the decay of maternal antibodies, while for others there was a statistically significant increase and others did not show differences . Notablyre lower . These Mre lower . The higre lower  showed t42 antibodies of all subclasses were lower in Manhiça than Kintampo  than in Manhiça (low MTI) for most antigens . At M0, Kintampo , 7. ThusKintampo  showed cKintampo .At baseline there were not very generalized or consistent significant associations with antibody levels and the rest of covariates in multivariable models . Hb conc42). Stratified by age, this was more significant in children , SSP2 and MSP1 bl2 constructs; and IgG4 to MSP1, VAR2CSA DBL1-2 and DBLα , while age (p = 0.76), site (p = 0.29), and vaccine (p = 0.57) were not. The predictive ability of the model was very high  with an AUC of 0.964.PLS-DA at M3 with malaria as a response variable  identifiand DBLα . In a mu1−4 subclass response.A summary of all results according to single-marker (univariable and multivariable regression models) and multi-marker (PLS-DA) analysis, is presented in P. falciparum antigens, whereas IgG2 responses were mostly associated with malaria risk. As expected, IgG1 followed by IgG3 were the predominant subclasses in most antigens, with lower IgG2 and IgG4 levels. Consistent with previous reports, MSP1 bl2, MSP2, and RH5 predominantly induced IgG3, or similarly high IgG3 and IgG1 levels, and we found that this was also the case for DBLα  were higher in infants than children and in malaria cases than controls. This could be explained by presence of maternal antibodies, which are preferentially of the IgG1 subclass due to the differential placental transfer  compared to the other subclasses (21 days) , 25, meaWe hypothesized that while cytophilic antibodies IgG1 and IgG3 would be associated with protection, non-cytophilic antibodies IgG2 and IgG4 would be associated with risk, as reported in other studies –15, 53, Plasmodium spp. as a means to shelter in phagocytes and then evade the immune system  decreased following RTS,S immunization, suggesting that RTS,S vaccinees were less exposed to the parasite due to vaccine efficacy. Importantly, among the antigens to which antibodies were higher after RTS,S vaccination, MSP1 bl2 Well, MSP2 FL Dd2, MSP5, and MSP6 showed higher levels in controls than in malaria cases and were associated with protection in adjusted models for IgG1 and some for IgG3, while IgG1 to AMA1 and RH1, which decreased or did not change with RTS,S vaccination, were associated with malaria risk. Similarly, higher M0 to M3 change of IgG3 levels to MSP1 bl2 and MSP5, which were increased by RTS,S vaccine, was observed in controls than malaria cases. In adjusted models, IgG3 to MSP1 bl2, MSP5  were also associated with protection at M3. In contrast to IgG1, stratified analysis including only RTS,S vaccinees showed that IgG3 to PE antigens LSA1 and SSP2 and MSP1 bl2, the latter two increased after RTS,S vaccination, correlated with protection. For IgG3, associations with malaria risk were only found for MSP2 FL CH150, which was not affected by RTS,S vaccination. In multi-marker analysis of M3 antibody levels, IgG1 levels to MSP142 and IgG1 and IgG3 levels to MSP2 contributed significantly and positively to the components associated with malaria, whereas IgG3 levels to numerous antigens including VAR2CSA DBL1-2, MSP1 bl2 3D7, MSP5 contributed to the components in an inverse relationship, consistent with the malaria protective associations in previous results. In addition, IgG3 to pTRAMP, CelTOS, RH4.9, SSP2, and RH2 were also important variables in the components suggesting a role in malaria protection. RTS,S vaccination also decreased the levels of IgG2 to EXP1, another exposure antigen, but it increased the levels of IgG2 to VAR2CSA DBL3-4 and IgG2 and IgG4 levels to MSP5. However, these responses were not associated with protection against malaria.Unexpectedly, IgG1 and IgG3 levels to some antigens  increased after RTS,S vaccination. In contrast, levels of IgG1 to exposure antigens  to protection. We wondered how RTS,S might benefit the response to other PE antigens and a protective effect in RTS,S vaccinees. We speculate that the anti-CSP antibodies induced by the vaccine prevent the quick invasion of the hepatocytes , and thiAge and site also affected IgG4 responses, children showing higher levels than infants, and Manhiça higher levels than Kintampo. In general, levels for IgG subclasses (less evident for IgG2) increased with age and IgG4 was not an exception. In fact, a previous study showed that IgG4 levels to EBA175 increased with age . As for 1−4 levels to MSP5 after RTS,S immunization and their association with protection  Institutional Ethics Committee (IEC), Ghana; Noguchi Memorial Institute for Medical Research IRB, Ghana; Comité Ètic d'Investigació Clínica , Spain; Comite Nacional de Bioetica (CNBS), Mozambique; Research Ethics Committee (REC), PATH, USA.CD, RS, and GM wrote the first draft. CD, JC, and GM conceived the study. RS, AA, IU, and CV performed database management, statistical analysis, and experimental design. AN, CJ, JC, GM, CD, DD, KA, BGy, and SO-A collected samples and data and participated in the clinical trial. MV, AJ, and IU performed the experiments. NW, ND-P, and CD coordinated the study. IU, CD, JC, GM, RA, and CV participated in the design of the analysis. DL, VC, CC, SD, DG, EA, BGa, RC, DC, JB, and LR provided antigens. JB contributed to the write up of the manuscript. All reviewed and approved the 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.


Plasmodium falciparum transmission over the last 15 years. Since immunity to malaria is acquired more rapidly at higher transmission, the slower acquisition of immunity at lower transmission may partially offset the benefits of reductions in transmission. We examined the clinical spectrum of disease and predictors of mortality after sustained changes in transmission intensity, using data collected from 1989 to 2016.Many parts of Africa have witnessed reductions in We conducted a temporal observational analysis of 18,000 children, aged 14 days to 14 years old, who were admitted to Kilifi County Hospital, Kenya, from 1989 to 2016 with malaria. We describe the trends over time of the clinical and laboratory criteria for severe malaria and associated risk of mortality.During the time periods 1989–2003, 2004–2008, and 2009–2016, Kilifi County Hospital admitted averages of 657, 310, and 174 cases of severe malaria per year including averages of 48, 14, and 12 malaria-associated deaths per year, respectively. The median ages in years of children admitted with cerebral malaria, severe anaemia, and malaria-associated mortality were 3.0  2.2–3.9), 1.1 (95% CI 0.9–1.4), and 1.1 (95% CI 0.3–2.2) in the year 1989, rising to 4.9 (95% CI 3.9–5.9), 3.8 (95% CI 2.5–7.1), and 5 (95% CI 3.3–6.3) in the year 2016. The ratio of children with cerebral malaria to severe anaemia rose from 1:2 before 2004 to 3:2 after 2009. Hyperparasitaemia was a risk factor for death after 2009 but not in earlier time periods.Despite the evidence of slower acquisition of immunity, continued reductions in the numbers of cases of severe malaria resulted in lower overall mortality. Our temporal data are limited to a single site, albeit potentially applicable to a secular trend present in many parts of Africa.The online version of this article (10.1186/s12916-019-1359-9) contains supplementary material, which is available to authorized users. Plasmodium falciparum malaria is an important cause of childhood morbidity and mortality in sub-Saharan Africa (sSA), which accounts for 90% of the world’s malaria deaths [Plasmodium falciparum transmission were observed from 2000 to 2010, but progress has stalled since 2010 [a deaths . Most maa deaths , 3. Globnce 2010  and the Immunity to clinical malaria is acquired following repeated exposure to malaria parasites. In high transmission areas, significant clinical malaria is rare after 5 years of age, owing to the acquisition of immunity in early childhood. By contrast, in low transmission areas, clinical malaria occurs among older children [The dominant severe malaria phenotype in areas of low transmission is cerebral malaria rather than severe anaemia . CerebraA decline in transmission occurred between 2000 and 2010 in many parts of Africa including the East African Coast . DetaileWe previously reported reduced mortality and morbidity during the period of declining malaria transmission in Kilifi County Hospital (KCH) in Coastal Kenya from 1989 to 2008 . HoweverSince May 1989, there has been continuous surveillance of hospital admissions to KCH as a partnership between the KEMRI-Wellcome Trust Research Programme and Kilifi County Department of Health. Consent for the use of data is sought from the parents or legal guardians of admitted children, and wider community engagement to explain research activity was undertaken . Linkage2 area, which included 202,000 individuals and 104,000 < 14-year-old children in 2002, rising to 287,000 individuals and 134,000 < 14-year-old children in 2016. The average age among the population of children below 14 years of age rose marginally from 6.7 years in 2002 to 7.0 years in 2016. No shifts in reported ethnicity are noted since 2002 to date. There are 27 major government primary health care facilities in the area in 2016, and in the absence of prospective monitoring data, we estimate that 7 of these are newly constructed in the last 15 years. User fees for healthcare access were introduced in Kenya in 1989 and removed in 2004, albeit with variable implementation and ongoing charges for inpatient care [The KHDSS covers an 891 kment care . There hThe pediatric service at KCH includes two wards; a 70-bed general ward and a 15-bed high dependency unit (HDU) staffed by research clinicians and nurses. The HDU admits children with serious illness requiring more intensive monitoring and management . Structured case records are completed electronically on all admissions, capturing age, residence, vital signs, clinical history and examination, and the Blantyre coma score . Data arP. falciparum malaria, we used the clinical criteria specified by the WHO [In defining severe  the WHO   excepting the three major grouping of severe malaria, age, and time period which were maintained regardless of significance; then adding interactions between covariates and time (excluding those for which p > 0.05). We also conducted a sub-analysis which included only children resident in the KHDSS, which afforded calculation of denominators to derive incidence rates.The analysis in this study runs from May 1989 to December 2016 and was restricted to children aged 14 days to 14 years of age. Stata software was used . The binomial method was used to calculate 95% confidence intervals for median ages, and Kruskal-Wallis to compare median ages by year restricted to subgroups of children with severe malaria. Logistic regression was used to examine risk of death, including all children admitted with a positive malaria slide. Three time-periods were defined: 1989–2003, 2004–2008, and 2009–2016, covering before, during, and after the reduction in malaria cases, respectively . In commBetween 6 May 1989 and 31 December 2016, there were 116,056 pediatric and neonatal admissions to Kilifi County Hospital (KCH), of whom 99,126 (85.4%) children were aged 14 days to 14 years. Within this age group, 17,691 (17.8%) presented with one or more criteria for severe malaria, of whom 12,805 (72.3%) had a parasite count of > 2500/μl. Annual pediatric admissions to KCH varied, from 3886 in the mid-1990s to a high of 4700 in the year 2000 before falling to 2986 in 2014 , 10 (16%), and 8 (18%) deaths in the periods 1989–2003, 2004–2008, and 2009–2016, respectively. The proportion of children with cerebral malaria gradually changed from 7% before 2004 to 22% after 2009 .In 1989, the median ages of children in the following groups (a) hospitalized with non-malarial conditions, (b) with severe malaria anaemia, and (c) with cerebral malaria were (a) 1.1 years (95% CI 1.0–1.2), (b) 1.1 years (95% CI 0.9–1.4), and (c) 3.0 years (95% CI 2.2–3.9), respectively. By the year 2016, the median ages had increased in each of these three group to (a) 1.8 years (95% CI 1.7–1.9), (b) 3.8 years (95% CI 2.5–7.1), and (c) 4.9 years (95% CI 3.9–5.9), respectively. Hence, although there was a slight increase in median age among children hospitalized with non-malarial conditions, there was a more marked increase in median ages in those hospitalized with severe anaemia and cerebral malaria  and 1.4 years (95% CI 0.9–1.8), respectively, rising by 2016 to 5 years (95% CI 3.3–6.3) and 1.2 years (95% CI 1–1.3), respectively. Similar results were seen using the alternate case definition , there was a slight, but statistically non-significant, increase in mortality on unadjusted analysis which was not evident after adjusting.p = 0.8), cerebral malaria (p = 0.7), age (p = 0.6), compensated shock (p = 0.7), and hypoglycaemia (p = 0.5) and respiratory distress (p = 0.7). Age and time period did not show a significant interaction (p = 0.7), indicating that the case fatality for a given age and phenotype was consistent over time. These interactions with time period were not retained in the final model. The adjusted interaction was statistically significant for hyperparasitaemia (p = 0.001), which was retained for the final interaction model  model, 804.7 for the multivariable (restricted) model and 803.6 for the interaction model with 13, 9, and 11 degrees of freedom, respectively. This indicates best fit with the interaction model.Over 27 years of continuous longitudinal surveillance, we show substantial changes in the numbers of children admitted to hospital with severe malaria. We previously reported the epidemiological transition to the nadir of severe malaria cases in 2009  and repoConsistent with previous reports comparing differing malaria transmission settings  or previIn longitudinal surveillance, the picture continues to evolve after a period of changing transition. For example, an area in transition from moderate to low will retain a cohort of older children who acquired immunity at moderate transmission intensity. Over time, a cohort of older children will emerge who acquired only limited immunity at low transmission intensity when they were younger, and as this cohort emerges, the average age of malaria will continue to rise and rates of admission with malaria may rise again after a nadir.What impact does lowered immunity have on case fatality? Predictors of case fatality remained constant over time, except for hyperparasitaemia which emerged as a risk factor for case fatality after 2009. The increasing proportion of children with cerebral malaria and the emergence of hyperparasitaemia as a risk factor might have been anticipated to lead to a relative increase in case fatality of severe malaria. There was a statistically non-significant increase in case fatality after 2009 in primary analysis Table , and a sCerebral malaria was associated with high case fatality throughout the monitoring period, as previously described . One of The study has limitations. Data were incomplete, particularly for the presence of multiple convulsions, although this was not an independent predictor of mortality. Secular trends may confound our analysis. Improved medical management has already been mentioned, and improved access to community and/or hospital healthcare is likely to impact the frequency and/or severity of presentation with malaria. While fertility rates are falling, increasing child survival and changes in hospital usage might have impacts on the median age of children coming to the hospital . There wPlasmodium falciparum histidine-rich protein 2 as a proxy for the sequestered parasite biomass [Defining the true burden of severe malaria is complex including the poor specificity of case definitions that include asymptomatic parasitaemia that coincide with a child presenting with severe illness due to another underlying non-malarial etiology . We miti biomass . Our def biomass  but coul biomass , 34 and  biomass , which w biomass , and we Our data indicate one possible outcome of a secular trend of reducing malaria transmission followed by stagnation, and this secular trend seems to be widespread in many parts of Africa . The traAdditional file 1:Table S1. Prevalence of asymptomatic parasitaemia among children admitted with trauma. (DOCX 12 kb)Additional file 2:Table S2. Frequency of mortality and criteria for severe malaria by Year. (DOCX 17 kb)Additional file 3:Table S3. Unadjusted logistic regression for risk of death by time period. Case definition includes parasite density threshold (i.e. > 2500 parasites per μl). (DOCX 14 kb)Additional file 4:Table S4. Unadjusted logistic regression for risk of death by time period. Case definition includes diagnosis by clinician. (DOCX 14 kb)Additional file 5:Table S5. Univariate and multivariate logistic regression models for risk of mortality in severe malaria. Case definition includes diagnosis by clinician. (DOCX 14 kb)Additional file 6:Figure S1. Numbers of admissions to Kilifi County Hospital over time. The percentage of admissions drawn from the Kilifi Health Demographic Surveillance System are shown on the left Y axis. Total annual admissions are shown on the right Y axis. The KHDSS was established in 2001, hence earlier residence in the DSS is reconstructed based on the reported location of residence in the clinical record. Hence the appearance of an expansion in KHDSS residence after 2001 is likely to reflect greater ascertainment of residence. (PNG 151 kb)Additional file 7:Figure S2. Trends in mortality, full clinical features of severe malaria and completeness of data collection over time. The trends over time are shown for clinical features of severe malaria (panels b-n) and mortality (panel a); giving the % of all admissions where a relevant observation for the panel subtitle was made ; the % of all admissions where the relevant observation was positive ; and the absolute number of cases where the observation was positive (blue line). (PNG 150 kb)Additional file 8:Figure S3. Trends in incidence of mortality and clinical features of severe malaria over time for Kilifi Health and Demographic Surveillance System Residents. The trends over time are shown for clinical features of severe malaria with a parasite threshold of > 2500 (panels a-e) and mortality (panel f); giving the % of all admissions where the relevant observation was positive ; and the incidence of cases per 1000 population among under 14 year old children (blue line). (PNG 124 kb)Additional file 9:Figure S4. Median ages for children admitted to Kilifi County Hospital. Case definition includes diagnosis by clinician. Median ages of presentation to hospital for specific phenotypes (see color legend) are shown over time with 95% confidence intervals calculated by the binomial exact method. (PNG 152 kb)Additional file 10:Figure S5. Parasite densities by year. The box plots show median parasite densities, interquartile ranges with boxes and adjacent values (i.e. lowest and highest observations within 1.5 times the interquartile range) with whiskers. Outlying values are shown by circles. (PNG 112 kb)


Despite substantial improvement in the control of malaria and decreased prevalence of malnutrition over the past two decades, both conditions remain heavy burdens that cause hundreds of thousands of deaths in children in resource-poor countries every year. Better understanding of the complex interactions between malaria and malnutrition is crucial for optimally targeting interventions where both conditions co-exist. This systematic review aimed to assess the evidence of the interplay between malaria and malnutrition.Database searches were conducted in PubMed, Global Health and Cochrane Libraries and articles published in English, French or Spanish between Jan 1980 and Feb 2018 were accessed and screened. The methodological quality of the included studies was assessed using the Newcastle-Ottawa Scale and the risk of bias across studies was assessed using the GRADE approach. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) guideline were followed.Of 2945 articles screened from databases, a total of 33 articles were identified looking at the association between malnutrition and risk of malaria and/or the impact of malnutrition in antimalarial treatment efficacy. Large methodological heterogeneity of studies precluded conducting meaningful aggregated data meta-analysis. Divergent results were reported on the effect of malnutrition on malaria risk. While no consistent association between risk of malaria and acute malnutrition was found, chronic malnutrition was relatively consistently associated with severity of malaria such as high-density parasitemia and anaemia. Furthermore, there is little information on the effect of malnutrition on therapeutic responses to artemisinin combination therapies (ACTs) and their pharmacokinetic properties in malnourished children in published literature.The evidence on the effect of malnutrition on malaria risk remains inconclusive. Further analyses using individual patient data could provide an important opportunity to better understand the variability observed in publications by standardising both malaria and nutritional metrics. Our findings highlight the need to improve our understanding of the pharmacodynamics and pharmacokinetics of ACTs in malnourished children. Further clarification on malaria-malnutrition interactions would also serve as a basis for designing future trials and provide an opportunity to optimise antimalarial treatment for this large, vulnerable and neglected population.CRD42017056934.PROSPERO The online version of this article (10.1186/s12916-018-1177-5) contains supplementary material, which is available to authorized users. Malaria and malnutrition in children  are reported independently to be major causes of morbidity and mortality in low- and middle-income countries. About 3.2 billion people remained at risk of malaria with an estimated 216 million new cases  and 445,000 deaths worldwide in 2016; the majority of deaths occur in children under 5 years in sub-Saharan Africa [Malnutrition is a complex phenomenon due to its multifactorial aetiology and diverse clinical presentation. Acute malnutrition manifests with wasting (low weight for height) and chronic malnutrition as stunting (low height for age). Being underweight (low weight for age) can result from either chronic or acute malnutrition or both. Assessment of malnourishment can be conducted using anthropometric indicators which compare child’s weight and height to the standardised age- and sex-specific growth reference derived from the international reference population of children between 6 and 59 months of age  Child Growth Standards 2006) . The antThe exact relationship between childhood malnutrition and malaria remains complex, controversial, and poorly understood. One of the key questions is, to what extent the burden of malaria is attributable to wasting and stunting? In regard to the impact of malaria on malnutrition, some evidence suggests malaria has adverse effects on nutritional status of young children –9. On thWe conducted a systematic literature review of manuscripts published between Jan 1, 1980, and Feb 19, 2018. PubMed, Global Health and Cochrane Libraries were searched using key terms  association between malnutrition and risk of malaria and (2) malnutrition and antimalarial treatment efficacy. We excluded studies primarily focused on non-malarial conditions such as TB, HIV, neglected tropical diseases, pneumonia or diarrhoeal diseases; non-clinical studies  and in vitro, animal, plant or molecular studies; studies on malaria in pregnancy and placental malaria; demographic and health surveys, mortality surveys, qualitative studies; case reports or series; vaccine studies; and studies primarily focused only on malaria or malnutrition or micronutrient deficiencies or anaemia. For this review, observational and interventional studies in non-pregnant populations with malnutrition assessed by anthropometric measurements as exposure and risk of malaria infection  as outcome were included.From each of the included studies in this review, the following variables were extracted: authors, year of publication, country, study design, age (range or median/mean), sex (ratio), sample size, growth standards, malaria transmission intensity, definition of malaria and the reported risk estimates. The methodological quality of the included studies was assessed using the Newcastle-Ottawa Scale (NOS)  and the P value was < 0.05 and/or the 95% confidence intervals (CIs) did not include 1. The risk of malaria was classified as “increased” or “decreased” according to the interpretation of the effect estimates provided (i.e. incidence risk ratio (IRR), odds ratio (OR), risk ratio (RR) or hazard ratio (HR)) if statistical significance was achieved as described above.Aggregated data meta-analysis was not possible due to the heterogeneity of studies in respect to study design, definition of malnutrition, definition of malaria, study population, e.g. age group target, analysis conducted and effect measures presented. Only summaries of study findings are presented in this review. Association between malnutrition and risk of malaria was deemed to be statistically significant if either the n = 16), longitudinal studies (n = 12), interventional studies (n = 3), case-control study (n = 1) and individual patient data meta-analysis (n = 1) , and significantly increased risk of any malaria parasitemia , high-density parasitemia  and severe anaemia  [falciparum malaria [A prospective cohort study of 487 children under 5 years of age in rural Gambia by Deen et al. reported that being stunted increased the risk of malaria infection significantly (RR = 1.35 (95% CI = 1.08–1.69)) . The aut 0.0001) . In cont 0.0001) . Two lon 0.0001) , 27. Sim 0.0001) . Verret  0.0001) . Contrar 0.0001) . This fi malaria .Eighteen studies explored the relationship between wasting and risk of malaria infection Table . OverallP. falciparum malaria in children with wasting. In a case-control study involving 428 under-five children in Ethiopia, Shikur and colleagues found that severely wasted children were three times more likely to have malaria episode than non-wasted children (adjusted OR = 2.90 (95% CI = 1.14–7.61) [P. falciparum infection was associated with wasting [P = 0.18) and high-density parasitemia , although increased the risk of severe malarial anaemia  [Takakura et al. , Ehrhard14–7.61) . Takakur wasting . However wasting . Similar = 0.04) . In cont = 0.04)  and in B = 0.04) , Eastern = 0.04)  and Ghan = 0.04)  reportedNineteen studies explored the relationship between being underweight-for-age and risk of malaria infection Table . OverallP = 0.007) in Cameroon. In a series of cross-sectional surveys conducted in the South Pacific island of Vanuatu in 1997, Williams et al. found a strong association between the incidence of P. vivax malaria and subsequently becoming underweight  but no significant effect of P. falciparum malaria  [In 2006, Ehrhardt et al. using cross-sectional surveys in Ghana found that being underweight was significantly associated with a higher risk of having fever of any cause , clinical malaria  and anaemia  . This wa.57–2.1) . On the .57–2.1) .Limited data exist on the effect of malnutrition on response to antimalarial drugs, in particular ACTs. Only five studies were identified in our literature search, and results were again contradictory.P = 0.046).In 2008, Obua et al. explored the impact of nutritional status on the dose, drug concentrations and treatment outcome with co-packaged chloroquine plus sulfadoxine-pyrimethamine in 83 children (6 months–5 years) with uncomplicated falciparum malaria . The autP. falciparum malaria, including in 43% chronically malnourished children [P = 0.022). Another study carried out in the DRC in 445 children, comparing the efficacy of standard doses of artesunate-amodiaquine between children with and without severe acute malnutrition (SAM), observed no evidence of reduced efficacy in children with SAM, which had an adequate clinical and parasitological cure rate, ACPR, of 91.4% [P = 0.038) [Pf malaria, the WorldWide Antimalarial Resistance Network (WWARN) demonstrated that among children 1–4 years of age in high-transmission areas, the risk of reinfection increased with a decrease in WAZ with a HR of 1.63 (95% CI 1.09 to 2.44) for a child with WAZ of − 3 compared to an adequately nourished child (WAZ = 0) [In a longitudinal study of 292 infants (aged 4–12 months) in Uganda, a high malaria transmission intensity setting, ACTs (artemether-lumefantrine and dihydroartemisinin-piperaquine) were generally efficacious with a good early parasitological response (99% of study participants cleared parasites by day 3) for treatment of children . Howeverof 91.4% . A recen= 0.038) . SimilarWAZ = 0) .P = 0.049). In the WWARN pooled analysis of individual PK-PD data from patients treated with artemether-lumefantrine for uncomplicated Pf malaria, underweight-for-age young children (< 3 years) had 23% (95% CI − 1 to 41%) lower day 7 lumefantrine concentrations than adequately nourished children of same age [Information on the pharmacokinetic properties of ACTs in malnourished children is critically lacking in the published literature. Our search retrieved a study published in 2016 which assessed the efficacy of AL in relation to drug exposure in children with SAM vs. non-SAM in Mali and Niger . This stsame age .The evidence on the effect of malnutrition on malaria risk remains controversial and in many instances contradictory. The current review highlights some key limitations in the way the interaction between malaria and malnutrition has been assessed and reported. First, differences in methodology, study populations, the variability in measures used to define malnutrition , and the heterogeneous malaria transmission intensities with different levels of host immunity within the different studies make the comparison challenging. Second, there is a paucity of information on the effect of malnutrition on therapeutic responses to ACTs and their pharmacokinetic properties in malnourished children in published literature. Generally, vulnerable populations with common co-morbidities such as malnutrition, obesity, HIV or tuberculosis co-infection are excluded from or under-represented in antimalarial drug efficacy trials . AlthougThis review has several limitations. First, one third of the studies included in this review recruited individuals of all ages, and disaggregating observations by the age of individuals (below and above 5 years) was not possible. Methodologically, the temporal relationship between malnutrition and risk of malaria  could not be assessed because of cross-sectional study design (50% included studies). It is also limited by the extent to which important confounders  are measured and reported in the included articles. Finally, the heterogeneity of the selected studies Additional file 2:PRISMA checklist (DOC 67 kb)Additional file 3:The methodological quality of the included studies (DOCX 33 kb)Additional file 4:N = 33) (XLSX 12 kb)Characteristics of the selected studies


Malaria is a major cause of anaemia in tropical areas. Malaria infection causes haemolysis of infected and uninfected erythrocytes and bone marrow dyserythropoiesis which compromises rapid recovery from anaemia. In areas of high malaria transmission malaria nearly all infants and young children, and many older children and adults have a reduced haemoglobin concentration as a result. In these areas severe life-threatening malarial anaemia requiring blood transfusion in young children is a major cause of hospital admission, particularly during the rainy season months when malaria transmission is highest. In severe malaria, the mortality rises steeply below an admission haemoglobin of 3 g/dL, but it also increases with higher haemoglobin concentrations approaching the normal range. In the management of severe malaria transfusion thresholds remain uncertain. Prevention of malaria by vector control, deployment of insecticide-treated bed nets, prompt and accurate diagnosis of illness and appropriate use of effective anti-malarial drugs substantially reduces the burden of anaemia in tropical countries. Plasmodium infect humans commonly, and all cause anaemia. Most malaria attributable deaths and severe disease are caused by Plasmodium falciparum. The majority of fatalities occur in the community. The World Health Organization (WHO) has estimated that there were some 216 million cases and 445,000 deaths from malaria in 2016 [Malaria is the most important parasitic disease of man . It is aThe clinical consequences of malaria, and in particular the prevalence of anaemia, depend on the intensity of malaria transmission Fig. . The maistable transmission. In the sub-Sahel region across Africa from Senegal to Sudan there is intense malaria transmission, but this is largely confined to the 3–4 month rainy season. During this period young children are commonly anaemic and frequently present to hospitals and health centres with severe anaemia. In contrast in areas where malaria transmission is low, erratic, or focal (often termed unstable transmission), protective immunity from malaria is not acquired, and symptomatic malaria may occur at all ages. In such areas changes in environmental, economic, or social conditions, such as heavy rains following drought or large population movements together with a breakdown in malaria control and prevention services (often resulting from conflict) can result in epidemics of malaria with considerable mortality among all age groups [Constant, frequent, year-round malaria reflects e groups . Recent P. falciparum and Plasmodium vivax) in pregnancy cause maternal anaemia, intrauterine growth retardation and prematurity [In areas of moderate or high transmission malaria infections . Malaria is usually associated with mild anaemia, although where resources are limited, then even at these low transmission intensities P. vivax may still cause severe anaemia in children because each sporozoite inoculation can result in multiple relapses. Transmission intensities are much higher in many parts of sub-Saharan Africa, where P. falciparum predominates, and in lowland New Guinea where both P. falciparum and P. vivax are prevalent  and, in some studies, these have been incriminated in causing red cell precursor apoptosis –44. It hSevere malarial anaemia in African children has been associated with the 238A TNF promoter genetic polymorphism and low levels of the anti-inflammatory cytokine IL-10 , 46. MalP. falciparum or by P. knowlesi the entire red cell population becomes less deformable [In severe malaria caused by formable –52. Whenformable  formable , 53, 54.formable .Fig. 5CoThe role of red cell membrane bound antibody (i.e. Coombs’-positive haemolysis) in malarial anaemia is unresolved. Some studies have shown increased red cell immunoglobulin binding in malaria, whereas others have not –58. In tIn acute malaria the spleen reorganizes and enlarges rapidly. This results in increased clearance capacity and a lowered splenic threshold for the clearance of abnormal erythrocytes, whether because of antibody coating or reduced deformability , 61–63. Repeated malaria infections result in splenomegaly and, in some cases, hypersplenism. At its most extreme is a condition called “hyperreactive malarial splenomegaly”, known in the past as “tropical splenomegaly”, in which there is massive splenomegaly, hypersplenism and dilutional anaemia. Some cases progress to B cell malignancy. Untreated the mortality is high, but when caused by malaria, the splenomegaly resolves over weeks or months with effective malaria chemoprophylaxis –73.The interaction between iron and malaria is complex and controversial. Iron deficiency is very common in malaria endemic areas. It causes anaemia and in young children iron deficiency is associated with neurodevelopmental delay. Malaria does not cause iron deficiency, but iron deficiency does reduce the incidence of severe malaria . NeverthPfHRP2) as the target antigen. PfHRP2 persists in pitted erythrocytes [pLDH-based tests become negative as parasitaemia clears. The RDTs for P. falciparum are slightly more sensitive than those for P. vivax malaria. Using sufficient volume blood samples PCR methods can now detect parasite densities 1000 times lower than microscopy or RDTs, but because of the high background rates of asymptomatic parasitaemia, even in low transmission settings [In the clinical assessment of anaemia a diagnosis of acute malaria requires either demonstration of malaria parasites in a thick or thin blood film, or a positive rapid test (RDT). Microscopy or RDTs have a detection threshold of approximately 50 parasites/μL, which also corresponds approximately with the pyrogenic density in non-immune subjects . The RDThrocytes  and so tsettings , they arsettings . HoweverAs the epidemiology of malaria coincides with the epidemiology of inherited red cell abnormalities, nutritional deficiencies and helminth infections, anaemia is often multifactorial, and the distribution of haemoglobin concentrations in healthy people is lower and broader than in temperate countries. Although a wide, and frankly confusing, variety of definitions of anaemia in general have been proposed, the most commonly used definitions in malaria studies, based on haemoglobin concentrations, are as follows.Mild anaemia ≤ 11 g/dLModerate anaemia ≤ 8 g/dLSevere anaemia ≤ 5 g/dL® system-a portable spectrophotometric analyser) or the haematocrit using a microcentrifuge. Well-functioning Coulter counters and other types of cell sorters are rarely found in rural areas of the tropics. The relationship between red cell count and haemoglobin or haematocrit is determined by red cell volume. In many areas microcytosis  is common. Malaria itself does not affect the relationship. The usual conversion factor of 3 for haemoglobin to haematocrit slightly overestimates the haematocrit [Pallor is readily recognizable clinically, and village health workers can be trained to recognize it, but anaemia is best quantitated by measurement in a capillary or venous blood sample of either the haemoglobin concentration , a good fit was obtained using Haematocrit = 5.62 + 2.60 * Haemoglobin .In areas of high malaria transmission where malaria is a major contributor to anaemia in the first years of life the ratio of haemoglobin to haematocrit changes with age . ClinicaThe term moderate anaemia has been used variably in epidemiological studies. For example, in a recent assessment of seasonal chemoprevention in malaria prevention moderate anaemia was defined as < 11 g/dL (and in this study severe anaemia was defined as < 6 g/dL) . In mostMalaria is an acute febrile illness. There are no specific clinical features in uncomplicated infections. Although, in general, higher parasitaemias are associated with more severe clinical disease, the relationship is very variable . In falcP. falciparum infections [Anaemia develops rapidly in acute malaria so the majority of symptomatic patients have already lost at least 1 g of haemoglobin per decilitre (100 mL) of blood before presenting to medical attention. The liver and spleen enlarge rapidly. The anaemia is haemolytic so red cell indices are usually normal, haptoglobin and haemopexin concentrations are reduced, and unconjugated bilirubin may be raised. The leukocyte count is usually in the low-normal range and the platelet count is nearly always reduced. Slight elevations in transaminases may occur—rises in aspartate aminotransferase  result both from haemolysis and liver injury whereas alanine aminotransferase  rises reflect liver injury only. With modern treatments (artemisinin-based combination treatments) defervescence occurs rapidly—and most patients have cleared fever and parasitaemia (assessed by microscopy) within 2 days . In highfections , 97, so Anaemia may be regarded a clock of the infection, in that a patient presenting with acute malaria and a normal haematocrit (provided they are not dehydrated) cannot have been ill for many days. The cumulative impact of repeated malaria episodes was documented in detail during the malaria therapy era, which began nearly a century ago, when malaria was used to treat neurosyphilis and also in detailed volunteer studies conducted by the military –102. In P. falciparum, although P. knowlesi and occasionally P. vivax may also cause severe disease [P. falciparum infection, but kidney injury, metabolic acidosis, and severe anaemia may occur in all the malarias [Severe malaria is usually caused by  disease . Severe malarias . The patmalarias . Anaemiamalarias . This hamalarias . In younSevere anaemia which may result either from repeated uncomplicated infections with any of the malaria parasites, or a more fulminant haemolytic anaemia which is usually associated with severe falciparum malaria or sometimes blackwater fever.Reduced microvascular perfusion resulting from cytoadherence and inter-erythrocytic adhesion.The consequences of severe anaemia are an appropriate increase in cardiac index to maintain oxygen delivery. Very severe anaemia ultimately leads to tissue ischaemia and hypoxia, with a rise in lactate production and an increase in the lactate-pyruvate ratio –106. TisThe first of these responds rapidly to blood transfusion.The relationship between haemoglobin concentration at presentation to hospital and outcome in falciparum malaria suggests that mortality rises sharply at levels below 3 g/dL (independent of other severe manifestations) , 110  mortalities range from 2.6 to 10.3%—while mortalities as low as 0.5% have been reported in children with severe anaemia but without any other severity manifestation . Those pAbove a haemoglobin concentration of 5 g/dL other manifestations are required for a diagnosis of “severe falciparum malaria”. In a very large series of over 8000 adults and children with strictly defined severe falciparum malaria studied in Asia and Africa over the past 37 years mortalities rose with increasing admission haemoglobin concentrations above 5 g/dL –117  which could be altered in severe malaria. Increasing haematocrit is associated with a linear increase in oxygen carriage but a non-linear increase in apparent viscosity. As a consequence there is an optimum haematocrit for oxygen delivery . The deps begins , 121. Fiear rate . The optear rate . Thus reAlternatively there is no causal relationship. Severe anaemia may simply reflect duration of illness, and thus control of a severe infection or series of infections without intervening lethal vital organ dysfunction. The observed relationship between admission haematocrit and outcome of severe malaria might be explained entirely by other covariate relationships. Clearly this is an important question requiring further study to inform treatment guidelines and patient management.This condition is well described but still poorly understood . Blackwaoi-RBC) have a much shorter survival time (7–14 days) compared with normal erythrocytes in healthy subjects (120 days) or in patients following severe malaria (44 days) [oi-RBC following artesunate probably reflects drug killing and then pitting of developed ring form parasites whereas background (and quinine associated) pitting may only occur for very young ring stages shortly after merozoite invasion with correspondingly less damage to the erythrocytes. The rapid and synchronous elimination of these oi-RBC from the circulation 1 to 2 weeks after the start of anti-malarial treatment results in haemolytic anaemia, which in some cases can be marked [There are several reports, mainly from temperate countries describing returned travellers, of late haemolytic anaemia occurring 1–3 weeks after parenteral artesunate treatment of hyperparasitaemic falciparum malaria. Most cases followed intravenous artesunate, although some were reported after intramuscular artemether, intrarectal artesunate and oral artemisinin derivatives. No deaths have been reported, but blood transfusion was often required. A recent review of published data, from non-immune and semi-immune patients, estimated the incidence of late haemolysis after intravenous artesunate to be 13% (95% CI 9–18%) and the requirement for a blood transfusion at 9% (95% CI 6–14%). Most of the data are from case reports and case definitions have varied substantially –69, 128.44 days) , 128. In44 days) . The shoe marked .Severe anaemia (haemoglobin < 5 g/dL) requires blood transfusion which can be life-saving , 130. ThThe anti-malarial treatment of choice for severe malaria is parenteral artesunate , 116. WhIn areas where the HIV prevalence is high severe anaemia with malaria is more likely in HIV positive patients , and in Blackwater fever results from massive haemolysis sufficient to cause haemoglobinuria. The management of blackwater fever anaemia is with blood transfusion . Anti-maP. vivax is endemic frequently recurring illness caused by repeated relapse is the main contributor to malarial anaemia in childhood [P. falciparum is endemic frequent infections or repeated treatment failure cause anaemia. The prevalence of anaemia therefore increases with transmission intensity, and where anti-malarial drug resistance compromises drug efficacy [A promptly treated discrete episode of malaria in a patient with a pre-morbid normal haemoglobin is unlikely to result in clinically significant anaemia. It is the cumulative impact of repeated illness from recurrent malaria that is the main cause. In areas where hildhood , 9. In aefficacy . In areaefficacy . Prompt efficacy , but preAnaemia is common in pregnancy in tropical regions. Malaria is a major contributor to anaemia in pregnancy , 14, 139