Plasmodium falciparum is one of the leading causes of human morbidity and mortality from infectious diseases, predominantly in tropical and sub-tropical countries. As genetic variations in the toll-like receptors (TLRs)-signalling pathway have been associated with either susceptibility or resistance to several infectious and inflammatory diseases, the supposition is that single nucleotide polymorphisms (SNPs) of TLR2, TLR4, TLR9, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and FCGR2A could modulate malaria susceptibility and severity.Malaria caused by This study was planned to make a further contribution to solving the problem of the real role of the most common polymorphisms of TLR4, TLR9, TIRAP and FCGR2A genes in modulating the risk of malaria and disease severity in children from Burundi, Central Africa. All the paediatric patients aged six months to 10 years admitted to the hospital of Kiremba, Burundi, between February 2011 and September 2011, for fever and suspicion of acute malaria were screened for malaria parasitaemia by light microscopy of thick and thin blood smears. In children with malaria and in uninfected controls enrolled during the study period in the same hospital, blood samples were obtained on filter paper and TLR4 Asp299Gly rs4986790, TLR9 G1174A rs352139, T-1486 C rs187084 TLR9 T-1237 C rs5743836, TIRAP Ser180Leu rs8177374 and the FCGR2A His131Arg rs1801274 polymorphisms were studied using an ABI PRISM 7900 HT Fast Real-time instrument.A total of 602 patients and 337 controls were enrolled. Among the malaria cases, 553 (91.9 %) were considered as suffering from uncomplicated and 49 (8.1 %) from severe malaria. TLR9 T1237C rs5743836CC was associated with an increased risk of developing malaria (p = 0.03), although it was found with the same frequency in uncomplicated and severe malaria cases. No other differences were found in all alleles studied and in genotype frequencies between malaria cases and uninfected controls as well as between uncomplicated and severe malaria cases.TLR9 T1237C seems to condition susceptibility to malaria in Burundian children but not its severity, whereas none of the assessed SNPs of TLR4, TIRAP and FCGR2A seem to influence susceptibility to malaria and disease severity in this population. Plasmodium falciparum is one of the leading causes of human morbidity and mortality from infectious diseases, predominantly in tropical and sub-tropical countries , circulatory collapse, respiratory distress, or haemoglobin level <5 g/dL. Otherwise healthy children seen in the hospital during the study period for minor surgery with a similar sex and age distribution and without asymptomatic parasitaemia according to the results of the screening with thick and thin films were enrolled as uninfected controls.All the paediatric patients aged six months to 10 years admitted to the hospital of Kiremba, Burundi, for fever and suspicion of acute malaria were screened for malaria parasitaemia by light microscopy of thick and thin blood smears, with two independent readers. If informed written or verbal (in case of illiteracy) consent was obtained from a parent or legal guardian, children for whom both readers were positive for the presence of criteria . In partBlood samples were obtained from all the children at enrolment, both cases and controls. Blood was collected on filter paper  for future DNA extraction and testing and kept in a cool, dry place. Samples collected were sent in the central laboratory in the Department of Maternal and Paediatric Sciences of the University of Milan, Italy, by airmail, once a month, for genetic studies.The blood spots on filter paper were cut into little fragments, then incubated for 1 hour in 3- mL lysis buffer at room temperature on an orbital shaker. Paper residues were separated from lysate by brief spinning. DNA from all samples was then extracted with a NucliSens EasyMAG instrument , using the Specific B protocol. The DNA extracted was quantified using a Nanodrop ND-1000 instrument , with elution buffer as blank. SNP genotyping was then performed on genomic DNA using pre-designed Taqman® SNP Genotyping assays  ,34. SNP X2) or the likelihood-ratio statistical test, as appropriate [Data were analysed using SAS for Windows v. 9.1 . Categorical variables are presented as numbers and percentages, and were analysed using contingency tables and the chi-squared or Fisher’s exact test, as appropriate. The Hardy-Weinberg equilibrium (HWE) was performed by comparing the numbers observed of different genotypes with those expected under the HWE for the estimated genotype frequencies and p > 0.05 was in HWE. Genotyping deviations from HWE were assessed under Pearson’s chi-square  and controls (6 % for TLR4 Asp299Gly and 2.4 % for TIRAP Ser180Leu). On the contrary, minor allele frequency for the other SNPs studied exceeded >20 % in all cases, reaching values around 40 % (42.3 %-45.2 %) and 50 % (43.0 %-48 %) for FCGR2A His131Arg and TLR9 G1174A, respectively. TLR9 T1237C rs5743836 C was found significantly more frequently in children with malaria than in uninfected controls (p = 0.04), although no difference was found between children with uncomplicated and those with severe malaria. No other significant difference among Burundian children with malaria and related controls was found as well as between children with uncomplicated and those with severe malaria for all the other allele frequencies studied .A total of 602 patients  and 337 uninfected controls  were enrolled. Among uninfected controls, 106 (31.4 %) were admitted for abdominal surgery, 79 (23.4 %) for orthopaedic surgery, 69 (20.5 %) for urogenital surgery, 55 (16.3 %) for plastic surgery and 28  were considered as suffering from uncomplicated malaria and 49 (8.1 %) from severe malaria . Table Table The overall data seem to indicate that none of the SNPs of TLR4, TIRAP and FCGR2A genes studied, assessed in Burundian children, play a role in modulating susceptibility to malaria and disease severity in this population. Regarding SNPs of TLR9, this study suggests that whereas no role may be ascribable to TLR9 G1174A and TLR9 1486 C, TLR9 T1237C seems to be associated with an increased risk of developing malaria, although this SNP is not associated with different disease severity. Despite data regarding association between polymorphisms and severity of the disease have to be evaluated with caution because the total number of severe cases, particular those with cerebral malaria, is too small to permit to draw definitive conclusions, these findings seem to indicate that variation in the characteristics of TLR9 can play a role in conditioning the development of malaria. Consequently, they can be useful in the identification of children at higher risk of developing the disease and those for whom the preventive measures are strongly needed. However, all of these data have to be confirmed with larger studies because in most of the cases data collected with this study are only partially in agreement with those that have been already published at this regard.et al. reported that this polymorphism was associated with an increased risk of severe paediatric malaria without affecting the risk of infection [et al. demonstrated that TLR4 Asp299Gly was more common in patients with low parasitaemia and concluded that TLR4 could have a genetic role in controlling the blood infection level in mild malaria and could indirectly suggest a protective effect of TLR4 Asp299Gly against severe disease [et al.[et al.[P. falciparum recognition and host response in humans and whether TLR4 could contribute to the control of infection.Data on TLR4 Asp299Gly are quite different from those reported by other authors who, on the other hand, obtained results largely conflicting with each other. Mockenhaupt nfection . On the  disease . Similare [et al. and Sam-l.[et al., respectl.[et al.-21. Conset al., studying children with cerebral malaria, found that patients with the C allele at -1237 and G allele at 1174 had higher levels of IFN-γ than those without these alleles [et al., despite reporting evidence of cis-variants acting on gene expression, did not find any convincing association between TLR9 SNPs and malaria severity [et al.[et al.[et al.[et al.[P. falciparum. Haemozoin is a dark-brown haem crystal produced by P. falciparum that functions as a carrier for P. falciparum DNA and functionally affects TLR9 response to parasite infection activating TLR9 proportionally to its concentration [Studies on TLR9 SNPs have also reported conflicting data. Sam-Agudu  alleles . Becauseseverity . The daty [et al. but contl.[et al., no assol.[et al. and Sam-l.[et al. could bentration . Unfortuet al.[et al.[et al.[et al.[In this study, contrary to what was reported by some authors ,25, no pet al. and Khorl.[et al. and by Hl.[et al.. These let al.[Regarding FCGR2A His131Arg, the data of this study are completely different from those reported by Schuldt et al. who founet al.,28-31. DP. falciparum infection have no influence in conditioning susceptibility to and severity of malaria in Burundian children. An exception might be represented by TLR9 T127C for which a significant correlation with an increased risk of developing malaria without any role in determining severity was found. This finding, when confirmed with further studies carried out with greater study samples, can permit to identify those subjects for whom preventive measure are strongly needed. Moreover, knowledge that same aspects of the innate immunity can have different efficacy in facing infectious agents can be of value in the preparation of specific vaccines when immune response evoked by the antigens included in the vaccines involves the factors with modified activity. This has to be considered also in the preparation of the developing malaria vaccines.Knowledge of the relationships between genetics, susceptibility to and severity of infections is essential to identify subjects at higher risk and to develop specific preventive measures. This is particularly import for malaria that remains one of the most important health problem in several geographic areas. This study shows that genetic polymorphisms of some factors involved in the innate immune response to CRP, C reactive protein; HWE, Hardy-Weinberg equilibrium; SNPs, single nucleotide polymorphisms; TIRAP, Toll-interleukin 1 receptor domain containing adaptor protein; TLRs, toll-like receptors.The authors declare that they have no competing interest. The study was partly supported by Bando Giovani Ricercatori 2007  and partly by ABM Onlus (that is a non profit organization).SE and NP designed the study and co-wrote the manuscript. CGM, AZ, CD, AS and LT carried out the laboratory assays. EB, AL, MS and MG visited patients and controls, collected the samples and entered the data in the database. MM interpreted results of genetic analyses. CP statistically analysed the data. All the authors read and approved the final manuscript.


Plasmodium falciparumhistidine-rich protein 2 (PfHRP2) are popular for diagnosis of this most virulent malaria infection. However, concerns have been raised about the longevity of the PfHRP2 antigenaemia following curative treatment in endemic regions.Effective diagnosis of malaria is a major component of case management. Rapid diagnostic tests (RDTs) based on PfHRP2 production and decay was developed to mimic the kinetics of PfHRP2 antigenaemia during infections. Data from two human infection studies was used to fit the model, and to investigate PfHRP2 kinetics. Four malaria RDTs were assessed in the laboratory to determine the minimum detectable concentration of PfHRP2.A model of PfHRP2 dynamics model indicated that in malaria naïve hosts, P. falciparum parasites of the 3D7 strain produce 1.4 × 10-13 g of PfHRP2 per parasite per replication cycle. The four RDTs had minimum detection thresholds between 6.9 and 27.8 ng/mL. Combining these detection thresholds with the kinetics of PfHRP2, it is predicted that as few as 8 parasites/μL may be required to maintain a positive RDT in a chronic infection.Fitting of the PfHRP2-based RDTs should be able to detect parasites on the first day of symptoms, and that the persistence of the antigen will cause the tests to remain positive for at least seven days after treatment. The duration of a positive test result following curative treatment is dependent on the duration and density of parasitaemia prior to treatment and the presence and affinity of anti-PfHRP2 antibodies.The results of the model indicate that good quality  Plasmodium falciparum causes the most severe form of malaria, and contributes to high morbidity and mortality in areas of the world where malaria is endemic. Accurate diagnostics are paramount for efficient treatment of falciparum malaria. In this context, the popularity of malaria rapid diagnostic tests (RDTs) has increased due to the WHO recommendation for parasitological confirmation of suspected malaria cases prior to treatment [P. falciparum specific RDTs function by detecting a water soluble protein produced by P. falciparum, namely histidine-rich protein 2 (PfHRP2). Detection is accomplished using monoclonal antibodies specific for PfHRP2. PfHRP2 is produced by the parasite throughout its asexual lifecycle; it is expressed on the surface of infected erythrocytes and released into the peripheral circulation during schizogony [PfHRP2-detecting RDTs have however been reported to have low specificity for diagnosis of current malaria infection in areas of high transmission [PfHRP2-detecting RDTs may return positive results when parasites are not present in the blood. This persistence also affects the suitability of PfHRP2-detecting RDTs for diagnosis of malaria in endemic regions where chronic asymptomatic infections are common. In addition, the timing of onset of detectable antigenaemia using the RDT, relative to the appearance of parasites in the bloodstream at a level detectable by standard microscopic examination of a thick blood film is not well defined.reatment . Comparehizogony . Due to smission  and follsmission ,6; that P. falciparum parasites sequester in splanchnic vascular beds during the last half of the asexual life-cycle they are not accessible for microscopic diagnosis at this time. An additional consequence of sequestration is that the total parasite biomass may be underestimated if peripheral blood sampling is relied upon. It has, therefore, been proposed that quantitative assays of PfHRP2 level provide a more accurate measurement of parasite biomass and potentially assist in determining the prognosis of severe malaria [As mature  malaria .PfHRP2 antigenaemia are not well understood, although key parameters such as rate of production and elimination half-life have been estimated from in vitro and in vivo studies [PfHRP2 is developed and used to assess its potential impact on the interpretation of malaria RDT results. This model was applied to clinical data to estimate circulating PfHRP2 levels, as well as the amount of PfHRP2 available for detection using RDTs that include a red blood cell (RBC) lysis step.The kinetics of  studies ,8. In thPfHRP2, incorporating both the production and clearance of the protein during a P. falciparum infection. In developing the model it was assumed that the host was infected with a single, relatively synchronous infection of P. falciparum with a two-day (48 hour) asexual life cycle.A mathematical model was developed to mimic the kinetics of P. falciparum indicate that PfHRP2 accumulates in the cell as the parasite develops within the erythrocyte with little membrane leakage, resulting in 78-99% of PfHRP2 being released into the circulation when the erythrocyte ruptures during schizogony [PfHPR2 occurred early in the asexual cycle, with all the protein remaining within the infected RBC (iRBC) until the time of schizont rupture when it is instantaneously released into the circulation. The number of parasites replicating (i.e. schizonts rupturing) at a given time during an infection was estimated from available clinical data. Where required, parasite density was converted to absolute number of parasites by multiplying the density by an assumed blood volume of 5 L (5 × 106 μL).Studies of in vitro culture of PfHRP2 distribution within the body were considered; 1) the minimum circulating concentration, estimated assuming that PfHRP2 is water-soluble and distributed in equilibrium throughout the extracellular water volume of 14 L [PfHPR2 is distributed only within the total blood volume of 5 L.Two scenarios for  of 14 L , 2) maxiPfHRP2 from the circulation was assumed to be continuous following a first-order decay process:The elimination of k dis the proportion of PfHRP2 remaining k days after it was produced . The exponent was based on the published PfHRP2 half-life of 3.67 days [where PfHRP2 (in grams) for any day during an infection (tH) was calculated using equation (2), while the minimum (tHmin) and maximum (tHmax) circulating concentrations of PfHRP2 (g/μL) were calculated according to equations (3) and (4), respectively.The absolute amount of t is the time in days with t = 1 being the first schizogony of the parasite, j pis the number of parasites within the host replicating on day j, and f is the amount of PfHRP2 produced per iRBC (g).where PfHRP2 measured by RDT or ELISA (t Tminand tTmax) exceeds t Hminand t Hmaxsince it contains the circulating PfHRP2 plus the PfHRP2 within the iRBCs which is released due to cell lysis as a consequence of an RDT or ELISA assay.The concentration of t qis the number of parasites within the host on day t (t q= t pon replication days in a synchronous infection).where PfHRP2 kinetics model was applied to two datasets.To fit the model and assess sensitivity, the Study 1. The first dataset was from a prospective, unblinded, Phase IIa clinical trial where volunteers were infected with blood stage P. falciparum [P. falciparum (3D7 strain) asexual parasites, and were given curative treatment soon after reaching the target parasitaemia of ≥ 1,000 parasites/mL, as determined by PCR quantification [PfHRP2 concentration were estimated from blood samples taken at pre-specified time points (approximately once or twice daily) from initial infection to parasite clearance.lciparum . The subfication . Detailsfication . For thePfHRP2, serial blood samples from each subject were assessed by PfHRP2 ELISA . To interpolate the amount of PfHRP2 present in the blood samples, a calibration curve was constructed using serial dilutions of a 3D7 P. falciparumculture supernatant and used as control standards in each ELISA. The concentration of PfHRP2 in this culture supernatant had previously been measured at 55.5 ng/mL by interpolating the ELISA optical density of serial dilutions against a stock of recombinant PfHRP2 protein with known concentration . PfHRP2 concentration within each sample was determined by interpolating the optical density of the sample with the standard curve using the software package Softmax Pro (Molecular Devices Inc.).To determine the concentration of circulating f parameter in the maximum concentration model. As the PfHRP2 quantification was conducted using RBC pellets, rather than the serum, only the maximum concentration model was considered during the model fitting. An initial value of f (0f) was assumed to be 5.2 × 10-15 g, as this was the median value previously reported for four P. falciparum isolates during in vitro culture [f (f*) was achieved by determining the multiplication factor (m) which produced the minimum residual sum of squares between the predicted PfHRP2 concentration and subject ELISA data such that 0mf* = f.The pre-treatment parasitaemia data was used to fit the  culture . CalibraStudy 2. The second dataset was a sample of three patients with neurosyphilis who had been treated by infection with P. falciparum [P. falciparum (blood-induced), whilst Patient S707 was reinfected with the McLendon strain of P. falciparum after being infected with the same strain 30 days prior [PfHRP2 antigenaemia in symptomatic, untreated infections.lciparum . Patientys prior . The parPfHRP2 concentration detectable by malaria RDTs four different RDT products were tested against serial dilutions of 3D7 parasite culture supernatant containing a known concentration of PfHRP2. This supernatant was the same used to generate the standard curve in the ELISA assay described for Study 1 above. The products tested were Firstsign™ - ParaView (Pan + Pf) Malaria Test , SD BIOLINE Malaria Ag Pf/Pv , Carestart™ Malaria HRP2/pLDH (Pf/PAN) COMBO  and ICT Malaria Combo Cassette Test .To assess the minimum PfHRP2 was added to the RDTs as per the manufacturer's instructions with the test scored as positive if the PfHRP2 band was visible and negative if there was no visible PfHRP2 band. The reader of the RDT was not blinded to the concentration of PfHRP2 added to the RDT. The maximum dilution producing a positive result was classified as the RDT detection threshold, and the concentration of PfHRP2 corresponding to this dilution was calculated.Each sample with a specified concentration of PfHRP2 model was calibrated using data from Study 1. The optimal individualized multiplication factor (m) for subjects varied between 17.5 and 39.4, suggesting that the amount of PfHRP2 produced by the 3D7 P. falciparum parasites was considerably higher than previously reported  in Study 2. The amount of circulating PfHRP2 (t Hminand tHmax), as well as the amount of PfHRP2 predicted to be measurable by ELISA (t Tminand tTmax) were calculated , the model was applied using the optimal value determined in Study 1  as well as the previously reported value of 5.2 × 10-15 g [PfHRP2 was predicted to be detectable was Day 2 for all three patients under the fitted model, and Day 4 or Day 7 under the unadjusted model for patients S561 and S811, and S707, respectively. For the fitted model, the number of days that PfHRP2 was predicted to be greater than 100 ng/mL ranged between 25 and 50 days under the minimum circulation model and between 29 and 56 days under the maximum circulation model. The duration during an infection when the PfHRP2 antigenaemia was above the nominated thresholds was always longer for the maximum circulation model compared to the minimum circulation model, and the model output was highly sensitive to changes in f. The fitted model always produced a longer period of antigenaemia above a threshold, and an equal or earlier transition to this state, than the unadjusted model, due to the higher production of PfHRP2 per parasite per replication cycle.The duration and first day that  10-15 g   and equilibrium parasite density (parasites/μL). Statistically significant models were achieved (P < 0.001) and are reported below. The values in brackets represent the SE of the coefficients.The equilibrium parasite density required to maintain the s Figure . ApplyinPfHRP2 above a nominated threshold. Parasite densities above the equilibrium would always produce PfHRP2 levels over the threshold. Fluctuating parasite densities, as may be seen in chronic infections, will also maintain PfHRP2 levels above the threshold provided the period of parasitaemia below the equilibrium value is short; the maximum duration of low parasitaemia will be dependent on the parasite dynamics and amount of PfHRP2 produced immediately prior to the drop in parasite density.These estimates of the equilibrium number of parasites represent the minimum number of parasites required consistently over time to maintain the level of PfHRP2 concentrations are shown in Table PfHRP2 dilutions . It has been previously reported that each P. falciparum parasite produces 5.2 fg  of PfHRP2 per cycle [PfHRP2 was predicted to be considerably lower than that actually measured in malaria naïve individuals infected with blood-stage 3D7 P. falciparum parasites. This difference may be a consequence of the differences between testing antigen production from cultures grown in vitro compared to in vivo measurements, a consequence of measurement error and stochastic variability, or the result of natural variation in protein production between different parasite strains [PfHRP2 should be minimal. Therefore, the two-fold variation between the calculated optimal values for the amount of PfHRP2 produced per parasite per replication cycle is likely a result of measurement error in the low parasite densities or ELISA assay, or stochastic variability in PfHRP2 production by individual parasites, possibly in response to host factors.A key parameter in modelling the kinetics of er cycle . However strains . The subPfHRP2 was assumed to be distributed throughout the extracellular water of the host and 2) a maximum concentration model in which the PfHRP2 was assumed to be restricted to the bloodstream only. It is highly likely that the actual distribution of PfHRP2 is somewhere between these two extremes so the values presented should be viewed in this context. For simplicity, it was assumed in the model that PfHRP2 was produced early in the asexual life cycle, although in vitro data indicate that PfHRP2 accumulates throughout the cycle [The model developed was applied under two assumptions: 1) a minimum concentration model in which the PfHRP2 ELISA kit could detect PfHRP2 concentrations down to 110 ng/L [PfHRP2 antigen, the diagnostic sensitivity of the tests varied between 6.9 μg/L and 27.7 μg/L of PfHRP2. The model output shows that for the fitted model, these values are achieved on, or immediately prior, to the first day of fever for the three patients in Study 2. In Study 1, the predicted amount of PfHRP2 would have been insufficient to produce a positive RDT test, as subjects were treated before becoming symptomatic, with peak parasite densities less than 10 parasites/μL. Field data indicates that PfHRP2-detecting RDTs can detect parasite densities less than 100 parasites/μL [PfHRP2 production as in the unadjusted model, or higher detection thresholds in the RDT may cause a delay in a positive RDT result.It was recently reported that a commercial 110 ng/L . In the sites/μL ,17. The PfHRP2 in natural infections. This replicates field data, where children with higher parasite densities at the time of treatment had a significantly longer antigen persistence compared to children with lower parasite densities [PfHRP2 in this circumstance is a likely underestimate of the expected duration of a detectable antigenaemia in symptomatic infection. Indeed, as few as eight parasites/μL may be required to maintain PfHRP2 equilibrium above 6.9 μg/L using the parameter space described.The model informs understanding on how duration of infection and parasite density impact on the longevity of ensities . In StudPfHRP2 is varied. The four products examined here have also been assessed as part of the WHO Malaria Rapid Diagnostic Test Programme where they were tested against wild-type isolates [P. falciparum parasites/μL, with overall positivity rates varying from 93.0% for Firstsign™ - ParaView (Pan + Pf) Malaria Test to 99.1% for Carestart™ Malaria HRP2/pLDH (Pf/PAN) COMBO. The results from this study are generally aligned with the product testing results, as the product with the lowest panel detection score, Firstsign™ - ParaView (Pan + Pf) Malaria Test, had the highest PfHRP2 detection threshold. The modelling results indicate that these tests should continue to perform consistently well at concentrations in the order of 50-100 parasites/μL, or better in some cases. This result reflects a previous study in which the ICT Malaria Combo Cassette Test was able to detect P. falciparum parasites below 100/μL in field samples [P. falciparum isolates at 200 parasites/μL [As has been reported before, the diagnostic sensitivity of malaria RDTs targeting isolates ,20. Each samples . Howeversites/μL , and in PfHRP2 antigen produced by parasites in natural human infection is available for measurement. However, it is possible that PfHRP2 binds or complexes with other ligands or proteins such as PfHRP2-specific antibodies, thereby reducing the amount available as free circulating antigen. In this respect, it is known that anti-PfHRP2 antibodies develop and may thereby act to reduce the amount of free PfHRP2 circulating [PfHRP2 contained within the iRBCs provided that the quantity of antibody in the serum is insufficient to bind all of the PfHRP2 released from the iRBCs before it reacts with the test band in the RDT. A caveat to this is that the parasites must be present in the blood sample tested by the RDT. Parasite sequestration can affect the availability of parasites in finger-prick samples, a factor which has not been considered in this model.The model and results presented in the current study assume all culating . While tPfHRP2-based RDTs that were tested should be able to detect parasites on the first day of symptoms, and that the persistence of the antigen will cause the tests to remain positive for at least seven days post-treatment. The post-treatment duration of positive tests is dependent on the duration and density of parasitaemia prior to treatment, and possibility other factors such as anti-PfHRP2 antibodies. In chronic infections a steady state parasitaemia of only 4-65 parasites/μL is likely to be sufficient to maintain a positive RDT result.Malaria RDTs are becoming the cornerstone of effective malaria diagnosis in primary healthcare settings. Understanding the benefits and limitations of these tools is important to guide clinicians and product development. The outputs of the mathematical model reported here indicate that the The authors declare that they have no competing interests.LM developed the model, conducted the analysis and drafted the manuscript. AB performed the laboratory testing of RDTs and ELISA assays. JMC participated in the design of the study. MLG participated in the design of the study, reviewed the model output and helped draft the manuscript. All authors read and approved the final manuscript.


Decreased nitric oxide (NO) and hypoargininemia are associated with severe falciparum malaria and may contribute to severe disease. Intravenous L-arginine increases endothelial NO in moderately-severe malaria (MSM) without adverse effects. The safety, efficacy and pharmacokinetics of L-arginine or other agents to improve NO bioavailability in severe malaria have not been assessed.In an open-label pilot study of L-arginine in adults with severe malaria (ARGISM-1 Study), patients were randomized to 12 g L-arginine hydrochloride or saline over 8 hours together with intravenous artesunate. Vital signs, selected biochemical measures (including blood lactate and L-arginine) and endothelial NO bioavailability  were assessed serially. Pharmacokinetic analyses of L-arginine concentrations were performed using NONMEM.Six patients received L-arginine and two saline infusions. There were no deaths in either group. There were no changes in mean systolic (SBP) and diastolic blood pressure (DBP) or other vital signs with L-arginine, although a transient but clinically unimportant mean maximal decrease in SBP of 14 mmHg was noted. No significant changes in mean potassium, glucose, bicarbonate, or pH were seen, with transient mean maximal increases in plasma potassium of 0.3 mmol/L, and mean maximal decreases in blood glucose of 0.8 mmol/L and bicarbonate of 2.3 mEq/L following L-arginine administration. There was no effect on lactate clearance or RH-PAT index. Pharmacokinetic modelling (n = 4) showed L-arginine concentrations 40% lower than predicted from models developed in MSM.In the first clinical trial of an adjunctive treatment aimed at increasing NO bioavailability in severe malaria, L-arginine infused at 12 g over 8 hours was safe, but did not improve lactate clearance or endothelial NO bioavailability. Future studies may require increased doses of L-arginine.ClinicalTrials.gov NTC00616304 The outcome of adults with severe malaria has improved with use of intravenous artesunate compared to quinine Km) of 100–150 µmol/L Km of CAT-1 in 75% of patients Nitric oxide (NO) bioavailability is reduced in both African children with cerebral malaria There have been no previous clinical studies of agents to improve NO bioavailability in severe malaria. Based on the safety, efficacy and pharmacokinetic findings in moderately severe malaria, we conducted a study of intravenous L-arginine infusion in Indonesian adults with severe malaria (ARGISM-1 Study). We evaluated the safety and efficacy profile, including the changes on vital signs, electrolytes and acid base status, lactate clearance and endothelial NO bioavailability, and performed a preliminary pharmacokinetic analysis.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Plasmodium falciparum parasitemia as used previously Patients from 18–60 years of age with severe malaria (SM) were enrolled. SM was defined as having ≥1 modified WHO severity criteria including i) acute renal failure (creatinine>265 µmol/L), ii) hyperbilirubinemia , or parasitemia of ≥100,000/µL), iii) blackwater fever, iv) hyperparasitemia (>10% parasitized red cells), v) cerebral malaria (Glasgow Coma Score<11), vi) hypoglycemia (glucose<2.5 mmol/L), vi) respiratory distress  was administered as a 12 g dose diluted to 10% in normal saline and given by an infusion pump over 8 hours, with a similar volume of normal saline being used in patients randomized to placebo. Patients had continuous electrocardiogram monitoring throughout and up to one hour after the end of the infusion. Vital signs including systolic (SBP) and diastolic blood pressure (DBP), respiratory rate (RR), pulse rate (PR), Glasgow Coma Score (GCS) and temperature were recorded at regular intervals during and after infusions. Oxygen saturation and blood methemoglobin concentrations were measured using a pulse co-oximeter . Hemoglobin and leukocyte counts were measured by Coulter counter at baseline and venous blood was drawn into lithium heparin tubes at 0, 1, 2, 4, 8, 9, 12 and 24 hours after the start of infusion. A hand held biochemical analyzer (i-STAT Corp) was used to measure venous potassium, chloride, bicarbonate and lactate at these time points Pre-specified criteria for cessation of the infusions were extravasation, a decrease in the SBP of >25 mmHg from baseline or to below 80 mmHg, venous potassium of ≥6.5 meq/L or ECG changes consistent with hyperkalemia, anaphylaxis, a venous blood glucose <3 mmol/L, bicarbonate <10 meq/L, pH <7.1 or methemoglobin level >12%.Venous blood was centrifuged within 20 minutes to obtain plasma and immediately stored at −70°C. Amino acids were extracted from 50 µl of plasma after the addition of 50 µl of an internal standard (norleucine) and 200 µl of cold ethanol. Deproteinized plasma was derivatized with AccQFluor reagent , and amino acids were measured by high-performance liquid chromatography  as previously described We used a model derived from a previous study of moderately severe malaria which was a two compartmental model with first order elimination Mean values of the vital signs and biochemical parameters before infusion were compared to mean values obtained during and after infusion using a paired t-test . If parameters were not normally distributed, values before and after infusion were compared by the Wilcoxon Signed Rank Test. Because analysis in this manner could mask transient but potentially clinically significant changes in a minority of subjects, we also analyzed the maximum change in the vital signs and biochemical markers at any time during and up to two hours after L-arginine infusion compared to baseline readings. This gave a “worst-case scenario”, however transient, for each marker. The mean of the pre-infusion values in each patient was subtracted from the lowest  and highest  measurement during infusion or in the two hours following infusion and expressed as mean maximum change. Relationships between maximum change and the dose weight ratio  were assessed by Spearman’s rank correlation coefficient (ρ). Sample size calculation estimated 20 patients should be randomized to L-arginine and 8 to saline to detect a 20 per cent difference in endothelial function. All analyses were performed with Stata 11 (Statacorp). A two sided value of p<0.05 was considered significant.st February 2008 to 28th February 2009; six received L-arginine and two, saline . Local circumstances, including difficulties in accessing the field site, prevented continuation of the study but did not affect the preceding conduct of the study. In patients who received L-arginine, four had one severity criterion , one had two criteria  and one had four severity criteria . In those who received saline, one patient had one severity criterion  and one had four criteria . All the patients were treated with intravenous artesunate and six received additional antibiotics. One patient with acute renal failure received peritoneal dialysis but went on to recover renal function, and there were no deaths in the study. The patients’ baseline details are shown in .Eight patients were enrolled in the study from 1, saline . Local cThere were no significant clinical adverse effects in any patient; in particular, there were no allergic reactions, convulsions, pain, rash, swelling or erythema at the infusion site. In the two patients who were not comatose, there was no increase in severity of nausea, vomiting, headache or prostration during or after the L-arginine infusion.) or saline. However, the mean maximum change in the systolic blood pressure during the infusion in those who received L-arginine was-14 mmHg (range −11 to −19 mmHg)  compared to −0.5 mmHg (range 0 to −1 mmHg) with saline; . The change in the mean maximal diastolic blood pressure in those receiving L-arginine was −12 mmHg, (range −6 to −16 mmHg)  compared to −6 mmHg (range −2 to −10 mmHg) with saline; There was no difference in coma recovery times between patients who received L-arginine (mean 35 hours [range 12–69 hours]) and those who received saline (34 and 40 hours). There was no significant effect on the mean pulse and respiratory rate before and after infusion of L-arginine and there were no notable changes on the electrocardiogram. There were no clinically significant changes in the mean systolic or diastolic blood pressure during and up to 16 hours after the end of the infusion in patients who received L-arginine  had elevated blood lactate (>2 mmol/L) at baseline, with the baseline lactate being higher in the two patients randomized to receive saline National changes in the implementation of pre-existing materials transfer agreements pertaining to sample export meant that serial measures of L-arginine could only be measured in the first five of the eight patients enrolled . These results represent a preliminary assessment of the pharmacokinetic behavior of L-arginine in patients with SM and the time-concentration curves for plasma L-arginine concentrations are shown in This is the first clinical trial of an adjunctive treatment aimed at increasing NO bioavailability in severe malaria. In this study L-arginine was given intravenously to adults with severe falciparum malaria at the dose of 12 g over 8 hours. There were no clinically significant adverse effects on symptoms, vital signs, selected biochemical measures and acid base status. No improvements in lactate clearance time or endothelial function were noted, though only 4 of 6 patients who received L-arginine had abnormal baseline values for lactate and endothelial function. Pharmacokinetic analysis of L-arginine demonstrated the clearance was approximately 40% higher in severe malaria, with concentrations lower than those predicted from a previous model developed in moderately severe malaria The effects of L-arginine in decreasing blood pressure are thought to be due to increased endothelial nitric oxide (NO) production Consistent with our previous study, there was no statistically significant change in the mean plasma potassium concentrations, but a transient and statistically significant but clinically unimportant mean maximal increase in potassium concentration in patients given L-arginine compared to pre-infusion levels. The mean maximal ranges of increase were 0.2 to 0.7 meq/L and no patient had a potassium concentration higher than 5 meq/L, including the patients with acute renal failure. This was comparable to increases previously seen in patients with moderately-severe malaria given 6 g over 30 minutes but lower than those given 12 g in the same time period The only commercially available intravenous L-arginine formulation is the hydrochloride form which contains 4.75 meq of hydrogen and chloride ions per gram. In MSM patients, there was a dose-dependent relationship between L-arginine with decreases in bicarbonate and pH, and an increase in chloride Km of the CAT-1 transporter in 70% of the patients We did not find any improvement in the lactate clearance time, and in contrast to our study in MSM, there was no increase in endothelial NO production The structural model derived from moderately severe malaria appears to describe the data in severe malaria accurately. Although the small numbers of patients in whom pharmacokinetic assessment was possible limited the ability to determine accurate estimates of pharmacokinetic parameters, typical population values of CL and V appeared to be ∼40% and ∼400% larger for patients with severe malaria compared to the previous study in MSM In summary, a 12 g dose of L-arginine infused at 1.5 g/hour for 8 hours in patients with SM was safe, well tolerated and resulted in statistically significant but transient and clinically insignificant effects on mean maximal change in systolic blood pressure, potassium, glucose, bicarbonate and chloride compared to pre-infusion levels. Although numbers were small and there were only modest baseline abnormalities in lactate concentrations and endothelial function, no improvement in lactate clearance or endothelial function were seen. Notably plasma L-arginine concentrations following infusion appeared to be 40 percent lower than predicted. Future studies in SM will require an increase in the dosage of L-arginine.Protocol S1Trial Protocol.(DOC)Click here for additional data file.Checklist S1CONSORT Checklist.(DOC)Click here for additional data file.


The association between placental malaria (PM) and first peripheral parasitaemias in early infancy was assessed in Tori Bossito, a rural area of Benin with a careful attention on transmission factors at an individual level.Statistical analysis was performed on 550 infants followed weekly from birth to 12 months. Malaria transmission was assessed by anopheles human landing catches every 6 weeks in 36 sampling houses and season defined by rainfall. Each child was located by GPS and assigned to the closest anopheles sampling house. Data were analysed by survival Cox models, stratified on the possession of insecticide-treated mosquito nets (ITNs) at enrolment.Among infants sleeping in a house with an ITN, PM was found to be highly associated to first malaria infections, after adjusting on season, number of anopheles, antenatal care (ANC) visits and maternal severe anaemia. Infants born from a malaria infected placenta had a 2.13 fold increased risk to present a first malaria infection than those born from a non infected placenta  when sleeping in a house with an ITN. The risk to present a first malaria infection was increased by 3.2 to 6.5, according to the level of anopheles exposure .First malaria infections in early childhood can be attributed simultaneously to both PM and high levels of exposure to infected anopheles. Protective measures as Intermittent Preventive Treatment during pregnancy (IPTp) and ITNs, targeted on both mothers and infants should be reinforced, as well as the research on new drugs and insecticides. In parallel, investigations on placental malaria have to be strengthened to better understand the mechanisms involved, and thus to protect adequately the infants high risk group. P. falciparum during pregnancy Factors influencing the time to first malaria infections in infancy were first investigated by Le Hesran et al. in 1997, who found an effect of placental infection by th month of pregnancy. On arrival at the maternity clinic for delivery, women were again given information on the study protocol. The informed consent written in French and in Fon was then submitted for approval. After delivery, women having had a stillbirth were excluded from the study.The study was conducted in the district of Tori Bossito located 40 km North-East of Cotonou, South Benin. The study area included 9 villages and 3 health centres  providing birth attendance and primary health care. A birth cohort was set up in June 2007. Recruitment was performed until July 2008. All women living in any of the nine villages, attending health centres for antenatal care and having no intention to move were proposed to enter in the study. Two months before the study started, study supervisors and community health workers visited all villages to meet the women and to inform them about the study. In addition, midwives were told to present the study to all women attending ANC visits from the 7For each delivered woman, personal data , use of Intermittent Preventive Treatment (IPTp) with Sulfadoxine-Pyrimethamine (SP) by the mother and obstetric history were asked at recruitment. Information on the possession of an insecticide-treated net (ITN) by the family at enrolment was collected, according to mother's declaration. A nutritional, entomological, climatological and biological follow-up was then started. At delivery, maternal blood and umbilical cord blood samples were taken to search for malaria infection and anaemia. Thick and thin placental smears were made to look for PM. At birth anthropometric measurements were performed ) with methods recommended by the WHO The active follow-up of the infants included several visits (weekly and monthly home visits) from birth up to 18 months . During P. falciparum infection. Anthropometric measurements  were performed every month from birth to 6 months, then at 9, 12, 15 and 18 months.Monthly visits included a systematic TBS to assess asymptomatic Mothers were also invited to bring their infants to the health centre at any time (passive follow-up), for free attendance in case of fever suspected by the mother or any clinical signs and then the same procedure  was applied. The overall prevalence of HIV is low (2%) in this area The protocol was approved by both the Beninese Ethical Committee of the Faculté des Sciences de la Santé (FSS) and the IRD Consultative Committee on Professional Conduct and Ethics (CCDE).Entomological data were collected during 2 years, using human landing catches performed every 6 weeks, 3 consecutive nights, in 36 sampling houses . To account for environmental factors which could influence transmission, climatic parameters  were recorded and rainfall was measured daily in each village. All infants were located by GPS and assigned to the closest anopheles sampling houses. The distance between infants and catching houses ranged between 0 and 3.1 km with a median at 373 m. Number of anopheles collected in sampling houses was thus available for each infant every 6 weeks.Plasmodium falciparum is the commonest species (95%), P. malariae and P. ovale representing respectively 3% and 2% P. malariae and parasitological results are given as Plasmodium sp. TBS performed on placental, maternal and cord blood at delivery were read within 24 hours. TBS performed on the occasion of monthly infant follow-up or during unscheduled consultations were not examined immediately. They were read twice several months later by two lab technicians in the Cotonou laboratory (with less than 1% of disagreement). They were made in the purpose of later confirmation of RDT results. All TBS were stained with Giemsa. Leukocytes and parasites were counted simultaneously. A TBS was declared negative if no parasite was found after 500 leucocytes had been counted. P. falciparum asexual blood forms were counted on Giemsa-stained smears. Haemoglobin rates were measured at birth and quarterly on blood samples with a Hemocue® analyser.All blood samples  performed on EDTA and placental smears were processed in Tori Bossito laboratory. Malaria is perennial in the study area and according to a recent entomological survey As all previous studies had shown an effect of placental infection during the first year of life, and as a full set of data was available only for the first 12 months of follow-up, we restricted our analysis to this period. First, baseline characteristics were compared between infants presenting at least one malaria infection vs no infection during the first year of life, using Pearson chi2 test or Fisher's exact test for categorical variables and Student t-test for quantitative variables. Then several survival analyses were performed.For survival analyse, first malaria infection  was defined as the first positive TBS, performed either at monthly home visits or when the child was brought to the health centre because of fever. Such consultations at the health centre could follow a weekly visit (active follow-up) or happen at any time if the mother suspected her child to be sick (passive follow-up).Two time-varying covariates were created to take into account factors influencing the exposure to malaria and its fluctuation over time: the season, defined as rainy or dry, according to the start and the end of the rains determined from rainfall measurements, and the number of anopheles collected in the GPS-located sampling houses, coded in 3 classes: 0, To assess the effect of PM on the time to first infection, Kaplan Meier curves and a log rank test were performed. Univariate Cox analysis was performed to study the association between all covariates and the first malaria infection and to obtain unadjusted hazard ratios (HR). The date of right censoring was the date of first malaria infection or the last available date of follow-up. Then, a multivariate analysis was performed to study the association between first malaria infection  and PM, adjusting on environmental and other covariates.Respect of proportional hazard assumption was assessed for all covariates by graphical methods (-Ln(Ln(S)) function of time) and by testing the interaction with time.Data were analysed with Stata v. 8.0  and SAS version 9.0 software .Data presented in this paper were collected from children recruited between June 4, 2007 and July 31, 2008 and followed during their 12 first months of life. Environmental data  used in this analysis were collected during 2 years, from June 2007 until August 2009.As presented in the flowchart diagram in PfEIR calculated on the two years duration of the study on all 9 villages indicates the existence of a slight but constant transmission in the area with an average of 20.5 infected anopheles/man/year and two peaks during the rainy seasons, consistent with another study performed in the area The Annual One hundred ninety-two infants had at least one episode of malaria infection. Thirty four percent (66/192) of malaria infections were detected during monthly visits and 66% (126/192) during unscheduled visits at the health facility for fever or history of fever. Among monthly visits, 12.1% (8/66) of the infants were feverish. Among unscheduled visits, 96.7% (119/123) of the infants were feverish and 76.4% (94/123) had a history of fever over the past 48 h.Factors associated with the occurrence of a first parasitaemia during the 12 first months of follow-up are presented in Six placental TBS were not available (not performed by midwives or laboratory mishandlings). All covariates directly or indirectly related to exposure such as spatial factors were strongly associated to higher proportions of infants with at least one infection. Proportions varied according to villages (p<0.001) and maternity clinics (p<0.001). Neither age nor gravidity was associated to first malaria infections, whereas malaria prevention measures  or the attendance to more than 3 antenatal care visits had a protective effect.Characteristics of infants had no effect on proportions of children presenting at least one malaria infection .TBS on cord blood was positive in 5 cases out of 604 samples (0.83%). These 5 infants were controlled at day 1 and no parasite was found in the peripheral blood TBS. Among these 5 infants, all were born from PM infected mothers and two of them further had a first malaria infection at one month and three months of age, whereas the others presented no detectable infection until the age of 12 months.The median time (range) of first malaria infection was 4.88 months (0.53–11.86) in infants born from infected placentas, while it was 6.11 months (0.53–12.15) in infants born from non infected placentas. Four point seven percent (9/192) of the infants presented a first infection at one month, 20.3% (39/192) before 3 months and 54.7% (105/192) before six months of age. In all time classes, these proportions were lower in the children with an ITN at home compared with the children without ITN (data not shown). For a better understanding of the occurrence of malaria in relation to the possession of an ITN, we calculated the proportions of infants who presented a first malaria infection according to PM infection and ITN possession . Among tResults are presented in All covariates respected the proportional hazard assumption, except the ITN covariate. Graphical methods showed that risks were not proportional with time and an interaction between ITN and age was found statistically significant (p = 0.04). Therefore, we decided to perform two separate analyses, according to the possession of an ITN or not. Results of the two analyses are presented in In the first group , the role of PM remained strongly significant in increasing the risk of a subsequent first malaria infection, after adjustment on exposure to anopheles, season, ANC visits and severe maternal anaemia. Infants with a history of PM had a 2.1 fold increased risk to present a first malaria infection compared to infants with no history of PM .Infants exposed to a high number of anopheles (>10 anopheles/3 days-collection) had a HR = 6.50, [3.56–11.84], p<0.001 to present a first malaria infection compared to infants not exposed to anopheles, at a given age. There was an increasing HR with the amount of exposure to anopheles, as the infants exposed to a low number of anopheles (1-10 anopheles/3 days-collection) had a HR = 3.20, [1.93–5.31], p<0.001 to present a first malaria infection compared to infants not exposed to anopheles.In the second group (i.e. not possessing an ITN (N = 183), first malaria infections were not statistically associated with PM at delivery with an HR = 1.18, [0.60–2.33], p = 0.62 and were borderline associated with an exposure to a low number of anopheles (p = 0.05).First malaria infections were borderline associated to the season, among infants in both groups (p = 0.06).The main result of our study is that first malaria infections in infants are associated with PM during pregnancy, and this effect persists after adjustment on environmental factors related to malaria transmission such as the exposure to anopheles bites or the season. It confirms the results of previous studies, while eliminating the possibility that PM could be only a proxy variable for malaria transmission. Indeed, the most logical alternative explanation for early infections in infants would be a massive exposure to malaria vectors, testified by a high rate of PM infections during the mothers' pregnancies.In our study, infants born from infected placentas (whatever the presence of ITNs in their homes) had a 1.6 fold increased risk to present a first infection than infants born from non infected placentas (p = 0.02). This result is consistent with the three main studies conducted previously in different parts of Africa, in Cameroon (1997), Tanzania (2005) and Gabon (2008) In the survival analyses, a significant relation was found between the risk of first infection and time-related environmental factors (anopheles catches and season). There was also a significant association between PM and the risk of first infection, particularly pronounced in infants whose mothers had declared to possess an ITN (Cox model), after adjusting for environmental covariates. The same association was not found in the non-ITN strata. The effect of ITNs on malaria transmission is certainly important. A Cochrane meta-analysis concerning areas of stable transmission (i.e. EIR>10 infective anopheles bites/man/year) comparable to Tori Bossito, estimated at 50% the ITN-attributable reduction in clinical episodes, and 13% the reduction of infection prevalence in childrenIn our study however, the information on ITN possession was collected by interview, and the entomologists involved in the study reported a limited use of ITNs by the population, so this variable should be interpreted with caution. The effect of ITNs deserves to be explored further, looking more carefully to environmental and behavioural factors, over a longer time of observation.We chose to use the season and the exposure to anopheles as proxies for malaria transmission for two main reasons. First, the seasonality in transmission has been shown to be logically related to the number of clinical malaria casesConcerning the detection of first malaria infections, we had to deal with various sources of information. A parasitaemia could be either found on the occasion of a monthly systematic visit including TBS, or after a weekly household visit by a community health worker, or following the mother's own-decision (supposedly because of a sick child) to attend the health centre. In the two latter cases, the parasitological finding would be guided by clinical signs (mainly fever) in the former a positive TBS could be found either in a symptomatic or asymptomatic context. In order to detect as many “first infections” as possible, we decided to merge all data, whatever their origin. There is a possibility that some asymptomatic parasitaemias could have been missed using such a procedure, but the only alternative way to detect all events would have been an active parasitological weekly follow-up for each child, with the risk of poor acceptance by the community. We chose not to increase the burden of biological samplings in children. Finally, it seems unlikely that an under-detection of some malarial events could have biased our results of association between PM and first parasitaemia. In addition, we think the use of Cox models taking into account the delay to event  instead of the calendar date has improved our statistical analysis.The absence of PCR testing on the whole cohort is probably a limitation of the study. On cord blood, it would have increased the sensitivity of the detection of congenital infections by 3 to 5-fold, according to previous studies On placental blood, PCR would have shown submiscroscopic infections, which might also have consequences on birth outcomes Contrary to previous studies Finally, first malaria infection in early childhood can be attributed simultaneously to both PM and high levels of exposure to infected anopheles. It emphasizes the importance of gestational malaria as a threat to the child's health, by increasing the risk of low birth weight, but also per se, by inducing a susceptibility to further malaria attacks. Protective measures as IPTp and ITNs, targeted on both mothers and infants should be reinforced, as stated by the Roll Back Malaria initiative in the Abudja declaration Table S1Baseline characteristics of infants with ≥1 malaria infection during the first 12 months of follow-up, Tori Bossito, Benin, 2007–2010. * Student t-test . £ Six placental malaria TBS were missing.(DOCX)Click here for additional data file.


Plasmodium vivax is increasingly recognized as a cause of complications and death. The features and prognosis of mixed Plasmodium species infections are poorly characterized. Detailed prospective studies that include accurate malaria diagnosis and detection of co-morbidities are lacking.Mortality from severe pediatric falciparum malaria appears low in Oceania but P. falciparum, 7.9% P. vivax, 14.7% P. falciparum/vivax) hospitalized over a 3-year period. Bacterial cultures were performed to identify co-incident sepsis. Clinical management was under national guidelines. Of 262 children with severe falciparum malaria, 30.9%, 24.8% and 23.2% had impaired consciousness, severe anemia, and metabolic acidosis/hyperlactatemia, respectively. Two (0.8%) presented with hypoglycemia, seven (2.7%) were discharged with neurologic impairment, and one child died (0.4%). The 27 severe vivax malaria cases presented with similar phenotypic features to the falciparum malaria cases but respiratory distress was five times more common (P = 0.001); one child died (3.7%). The 50 children with P. falciparum/vivax infections shared phenotypic features of mono-species infections, but were more likely to present in deep coma and had the highest mortality . Overall, bacterial cultures were positive in only two non-fatal cases. 83.6% of the children had alpha-thalassemia trait and seven with coma/impaired consciousness had South Asian ovalocytosis (SAO).We followed 340 Papua New Guinean (PNG) children with PCR-confirmed severe malaria (77.1% P. falciparum/vivax infections were associated with the most severe phenotype and worst prognosis.The low mortality from severe falciparum malaria in PNG children may reflect protective genetic factors other than alpha-thalassemia trait/SAO, good nutrition, and/or infrequent co-incident sepsis. Severe vivax malaria had similar features but severe  Plasmodium falciparum infections in children is relatively low in the Oceania region P. vivax in early childhood. Nevertheless, P. vivax is itself increasingly recognized as a cause of both complications similar to those seen in falciparum malaria and death Plasmodium species infections , there are reports of reduced P. falciparum mono-infections.The features and prognosis of severe pediatric falciparum malaria have been characterized in observational and intervention studies from sub-Saharan Africa The incidence of complications and death from severe malaria will depend on selection/recruitment strategies, sensitivity of malaria diagnosis/speciation, definitions of severity, inpatient management including identification of co-incident disease, and social, cultural and genetic factors that can influence presentation and clinical course. Apparent inconsistencies between published studies may reflect differences in one or more such factors. The characteristics and outcome of severe malaria are, therefore, best assessed prospectively, in a representative, ethnically homogeneous sample, and with clinical and laboratory data sufficient both to allow accurate diagnosis P. vivax and mixed P. falciparum/vivax malaria, although less common than that caused by P. falciparum, have a similar spectrum of clinical disease and prognosis.We have carried out such a study in children in coastal Papua New Guinea (PNG) presenting with severe malarial illness, a setting in which a high prevalence of alpha-thalassemia and South Asian ovalocytosis (SAO) implies that malaria has had a strong selective effect P. falciparum and 24 for P. vivaxP. falciparum and 12 for P. vivaxP. falciparum (median [interquartile range (IQR)] 1360 [453–2881]/µL) and 14.1% (348 [226–727]/µL) for P. vivaxThe present study was conducted in Madang and Sepik Provinces on the northern PNG coast where most of the population are subsistence farmers and their families. The annual entomological inoculation rate (EIR) for Madang Province has recently been estimated at 37 for The present study was approved by the PNG Institute of Medical Research Institutional Review Board and the Medical Research Advisory Committee of the PNG Health Department, and conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from parent(s)/guardian(s) before recruitment.Plasmodium species were detected by nested polymerase chain reaction (nPCR) were included in the present analyses. The final speciation as a mono- or mixed infection was based on the nPCR result.Between October 2006 and December 2009, all children aged 0.5–10 years admitted to Modilon Hospital, the provincial hospital to which the majority of children with severe illness are referred, were assessed for recruitment to an observational study of severe pediatric illness. Inclusion criteria included any of: i) impaired consciousness/coma (Blantyre Coma Score (BCS)<5 After recruitment, a standardized case report form that recorded demographic and medical data was completed A BCS≤2 was considered deep coma and a BCS≤4 as impaired consciousness at 0.5, 1 or 6 hours after correction of hypoglycemia, a seizure or parenteral anticonvulsant therapy, respectively. Respiratory distress was considered present if the child had i) deep breathing, ii) inter-costal in-drawing, iii) sub-costal recession, iv) persistent alar flaring, v) tracheal tug, and/or vi) respiratory rate>60/minute. Due to variable availability and safety concerns, chest radiography was performed in a minority of children with respiratory symptoms. Inpatient management, including the decision to perform lumbar puncture (LP), was co-ordinated by attending ward clinicians under PNG national guidelines A baseline venous blood sample was taken for microscopy and rapid diagnostic testing (RDT) . Giemsa-stained thick blood smears were examined and the parasitemia quantified independently by two skilled microscopists with discrepancies adjudicated by a third microscopist 3 were present, CSF was incubated on chocolate agar for 3 days. Bacterial isolates from blood/CSF cultures were identified using standard procedures Corynebacterium and Bacillus spp. were considered contaminants. The remaining baseline blood sample was centrifuged promptly and separated plasma and erythrocytes stored at −70°C and −20°C, respectively.When sufficient blood was available from the admission venepuncture after the above tests, 1–3 mL was placed in Bactec™ Peds Plus™/F bottles  and incubated using an automated Bactec™ system. Cerebrospinal fluid (CSF) obtained at LP was examined macroscopically and microscopically Plasmodium speciation by nPCR Additional laboratory tests not necessary for initial clinical management were performed subsequently and included i) plasma electrolytes, urea, creatinine, alanine aminotransferase (ALT), total bilirubin, C-reactive protein (CRP) and creatine kinase , ii) Reference intervals for laboratory tests were determined from 327 healthy Melanesian children who were age- and sex-matched to the present children Plasmodium species with those of community-acquired malaria in a similar epidemiologic situation, we used data from 264 children aged 1–4 years from the Ilaita area of neighbouring East Sepik Province who were followed with fortnightly active morbidity assessment and passive case detection at a local health center between April 2006 and August 2007 To compare the proportions of the present hospitalized cases by post hoc two-group comparisons by Dunn's test for continuous and Bonferroni's correction for categorical variables. Crude odds ratios (ORs) for the observed frequencies of overlapping clinical phenotypes were calculated using 2×2 contingency tables. Multivariate analysis was performed using backward stepwise logistic regression. Variables other than age were entered based on biological plausibility and P<0.20 on univariate regression analysis and the most parsimonious model chosen using Aikake's Information Criterion. A two-tailed significance level of P<0.05 was used throughout.Summary data are presented as median and [IQR]. Comparisons of variables between groups were by parametric or non-parametric tests as appropriate, with Plasmodium DNA was undetectable by nPCR. The remaining 340 children had a median age of 40 [29–55] months and 189 (55.6%) were males. In most cases (87.1%), both parents were from Madang or East Sepik provinces, with the rest from other parts of PNG. Based on clinical features, blood film microscopy and nPCR, there were 262 severely-ill children with P. falciparum (77.1%), 27 with P. vivax (7.9%), 50 with mixed P. falciparum/vivax (14.7%) and one with mixed P. falciparum/malariae  hospitalized children aged between 6 months and 10 years were screened for inclusion to the study see . Of 353 P. falciparum mono-infections  but not in children with P. vivax mono-infections. For P. falciparum mono-infections by nPCR, there was 98.5% concordance with microscopy; two cases were diagnosed as P. vivax and two as P. falciparum/vivax. For P. vivax mono-infections by nPCR, there was 88.9% concordance with microscopy; three cases were diagnosed as P. falciparum mono-infections. Six of these seven discordant results were in patients with low parasite densities (<220/µL). For mixed P. falciparum/vivax infections, concordance was only 16%, with 68% diagnosed as P. falciparum and 16% as P. vivax mono-infections by microscopy.Of the sample of 340 children, 19.5%, 5.3% and 5.3%, respectively, had been treated with oral, parenteral or oral plus parenteral antimalarial therapy before admission. Prior treatment was associated with a lower parasite density in children with P. falciparum mono-infections, 81 (30.9%), 65 (24.8%) and 61 (23.2%) had impaired consciousness, severe anemia, and metabolic acidosis/hyper-lactatemia, respectively (see P = 0.005) and a greater than expected frequency of impaired consciousness plus acidosis (OR 2.1 (1.1–3.7), P = 0.02). Severe anemia plus acidosis occurred at expected frequency (OR 1.2 (0.6–1.9), P = 0.61). There were 7 children with all three phenotypes. Only two had hypoglycemia on admission.Of 262 children with severe vely see . There wP = 0.001). There were no other statistically significant differences between the presenting features of severe malaria caused by P. vivax or P. falciparum.Eight children with severe vivax malaria (29.6%) presented with impaired consciousness or deep coma, three (11.1%) with severe anemia, and five (18.5%) with metabolic acidosis or hyperlactatemia. Children with severe vivax malaria had higher median hemoglobin concentrations, smaller spleens, higher median plasma sodium concentrations, and lower median plasma bilirubin and CRP concentrations than children with severe falciparum malaria see . After aP. falciparum/vivax had a higher median respiratory rate (P = 0.014), a lower BCS (P = 0.018) and a greater proportion with deep coma  with contaminants identified in 13 (5.0%). Only two significant isolates were obtained, a non-albicans Candida from an admission blood culture from an immuno-competent child and a Klebsiella pneumoniae from a child after a week of inpatient treatment for severe malaria. Both children were treated with appropriate antimicrobial therapy and discharged well. A LP was performed in 129 children of whom 11 had>10 white blood cells/µL CSF. Routine bacterial culture, bacterial antigen testing, and India ink staining for Cryptococcus gattii, were negative in all cases.Most children responded to antimalarial/supportive therapy and were discharged well, including the two hypoglycemic children and the child with P. vivax case was a previously well child who presented with breathlessness, and abdominal/lower limb swelling. She had severe respiratory distress, a raised jugular venous pressure and an oxygen saturation of 92% despite supplementary oxygen. Her hemoglobin and blood lactate were 41 g/L and 2.7 mmol/L, respectively. A clinical diagnosis of pericardial effusion with cardiac tamponade was made and she had a cardio-respiratory arrest during attempted pericardiocentesis. Subsequent biochemical analysis revealed renal failure (serum creatinine 460 µmol/L), acidosis and hyperkalemia. The children with mixed P. falciparum/vivax infections had the greatest mortality . Of these four deaths, three were due to cerebral malaria. The fourth child had a hemoglobin concentration of 19 g/L and suffered a cardio-respiratory arrest before blood transfusion could be arranged.Six children (1.8%) died - one with falciparum (0.4%), one with vivax (3.7%) and four with mixed-species infection (8.0%) - all within 12 hours of admission. The child with falciparum malaria presented deeply comatose and died of cardio-respiratory failure. The fatal P. falciparum and one with P. falciparum/vivax, were discharged with neurologic impairment; three with deep coma on admission developed cortical blindness and five had motor deficits ranging from mild ataxia to spastic quadriparesis. Only two of these eight children had persistent neurologic deficits at follow-up two months later. Another child who recovered from severe vivax malaria with respiratory distress died between discharge and follow-up. When admitted, he had clinical signs of severe pulmonary hypertension and a prior history of echocardiographically confirmed cyanotic heart disease.Eight children (2.4%), seven with P. vivax, mixed P. falciparum/vivax and P. falciparum, respectively. Three children with SAO deletions presented with deep coma  and a further four with impaired consciousness. At least one alpha-thalassemia deletion was observed in 83.6% of the children with severe malaria (see The genetic deletion causing SAO was present in 17 of 320 (5.3%) children, including 1, 2 and 14 with severe disease due to aria see .P. falciparum/vivax infection is similar to that with P. falciparum alone  but greater than with P. vivax alone . The main manifestations of severe malaria in the 30 children were severe anemia (46.7%) and multiple convulsions (36.7%). Two children with severe falciparum malaria died, one from cardio-respiratory failure secondary to severe anemia (hemoglobin 16 g/L). The other was an anemic child (hemoglobin 63 g/L) with P. falciparum whose condition deteriorated after outpatient antimalarial treatment and who died 6 hours later.Episodes of malaria during a total of 247 person-years of follow-up in the East Sepik cohort study P. falciparum malaria in Melanesian children presents with the same phenotypes as those in African children, the mortality is low (<1%) in a hospital setting. The relatively few cases of severe vivax malaria in our series exhibited presenting features that were similar to those in the children with severe P. falciparum malaria but with a higher likelihood of respiratory distress. Severe mixed P. falciparum/vivax infections were greater in number than the vivax malaria cases. These children also had comparable admission clinical/laboratory findings to those of the patients with severe falciparum and vivax malaria, but their subsequent mortality was the highest of the three groups. These observations confirm and extend data relating to severe malaria in an area of intense transmission of multiple Plasmodium species, especially in relation to mixed-species infections.The present study confirms that, although severe Our children with severe falciparum malaria had rates of impaired consciousness, acidosis, anemia, and combinations of these, that were similar to those reported in African studies Case fatality rates in African children with severe falciparum malaria lie between 3% and 50% Salmonellae infection that substantially increase mortality Salmonellae were isolated from the present or other children with severe non-malarial disease studied contemporaneously (data not shown). All our children were treated empirically with intramuscular artemether, parenteral chloramphenicol and other supportive therapy African studies show hypoglycemia-associated mortality of >50% Plasmodium speciation using molecular methods.Severe vivax malaria has been associated with a wide variety of clinical manifestations in adults and children that include severe anemia, altered consciousness, respiratory distress, jaundice, thrombocytopenia, acidosis and renal failure P. vivax infection from the island of New Guinea P. vivax malaria. In the second study, which involved prospective community-based morbidity surveillance P. vivax cases than those with P. falciparum. Acute respiratory distress syndrome (ARDS), interstitial pneumonia and pulmonary oedema can complicate vivax malaria in adults P. vivax-infected erythrocytes can bind to cells expressing endothelial receptors known to mediate the cytoadhesion of P. falciparumA case in point is respiratory distress. In one of two recent prospective studies of severe pediatric P. vivax malaria in our cohort. Although this child died during attempted pericardiocentesis, she had severe anemia, respiratory distress, renal impairment and acidosis. In the larger observational study from southern Papua P. falciparum or vivax) was lower in children than adults but no pediatric-specific vivax malaria mortality data were reported in this study or a previous prospective observational study from PNG There was a single death from P. falciparum/vivax infections had clinical and laboratory features that were mostly similar to those of the severe falciparum cases despite generally lower P. falciparum parasitemias. The exceptions were a higher baseline respiratory rate and a lower BCS. These features suggest that the children in this group were the most severely ill, at risk of the pathophysiologic effects of both parasite species with the possibility that P. vivax microvascular cytoadherence as observed in lung Our children with severe mixed P. falciparum/vivax infections than severe falciparum or vivax malaria P. vivax infections to be associated with respiratory distress Plasmodium species. Nevertheless, our data indicate that WHO criteria for severe malaria P. falciparum encompass the spectrum of severe infections caused by P. vivax with or without co-incident P. falciparum.Other studies have found that, in contrast to the present data, severe anemia is less common in severe P. falciparum mono-infections and P. falciparum/vivax infections in a PNG child are more likely to progress to severe disease than P. vivax mono-infections. Indeed, the numbers of severe cases in these categories  were consistent those of the present study , suggesting that our hospitalized patients were representative of malaria in the community. The mortality in our series was greatest in severe P. falciparum/vivax infections, consistent with the presenting clinical and laboratory features of these children. In the two other studies with mortality data Although from a study with a different design, the community-based longitudinal data from a cohort of similarly aged East Sepik children who had similar red-cell polymorphisms and who were from an area of comparable malaria transmission post mortem tissue samples that may have clarified underlying pathology is culturally problematic in PNG.Our study had limitations. It is possible that the most severely ill children in the study catchment area die before they can be transferred for inpatient care, thus compromising the representative nature of our sample. Due mainly to issues with obtaining a sufficient volume of blood from a small, distressed and severely ill child, we could not exclude bacteremia in every case, while diagnostic tests for viral and fungal pathogens were not possible in this clinical setting. Obtaining P. vivax densities that can be missed by microscopy in this situation probably reflecting density dependent cross-species regulation The strengths of the present study were its prospective standardized data collection, the use of nPCR as a way of compensating for the known limitations of microscopy, and the availability of bacteriology facilities that enabled detection of common invasive bacterial infections. Such a rigorous approach has been used in few previous descriptive studies of severe childhood malaria. Our data confirm the lack of sensitivity of microscopy in identifying mixed species infections Plasmodium species has identified significant differences between the presentation and outcome of severe P. falciparum, vivax and falciparum/vivax infections in PNG. The low mortality observed for severe falciparum malaria does not appear related to high endemicity of P. vivax but may reflect protective genetic factors, better prior nutrition, and/or less exposure/susceptibility to secondary bacterial infection than in African children. Severe vivax malaria presents with features seen in severe P. falciparum infections but respiratory distress is more prominent, suggesting preferential cytoadherence of this parasite within the lung microvasculature. Severe P. falciparum/vivax infections in PNG children appear to have the worst prognosis, perhaps because they share the adverse presenting features of the respective mono-infections. These findings provide impetus for further research on the pathogenic potential of P. vivax infections when this parasite is present alone and especially in combination with other Plasmodium species.The present study of well-characterized children from an area of intense transmission of multiple


Plasmodium falciparum exposure, based on limited epidemiological data and short-lived antibody responses in some longitudinal studies in endemic areas.Malaria immunity is commonly believed to wane in the absence of 42 (3D7 and FVO strains), EBA-175 and DBL-α were determined by Luminex. IgG levels against antigens on the surface of infected erythrocytes (IEs) were measured by flow cytometry.A cross-sectional study was conducted among sub-Saharan African adults residing in Spain for 1 up to 38 years (immigrants) with clinical malaria (n=55) or without malaria (n=37), naïve adults (travelers) with a first clinical malaria episode (n=20) and life-long malaria exposed adults from Mozambique (semi-immune adults) without malaria (n=27) or with clinical malaria (n=50). Blood samples were collected and IgG levels against the erythrocytic antigens AMA-1 and MSP-142 (P≤0.015), similar levels against EBA-175 and DBL-α, and lower levels against IEs (P≤0.016). Immigrants had higher IgG levels against all antigens tested compared to travelers (P≤0.001), both with malaria.Immigrants without malaria had lower IgG levels than healthy semi-immune adults regardless of the antigen tested (P≤0.026), but no correlation was found between IgG levels and time since migration. Upon reinfection, immigrants with malaria had higher levels of IgG against all antigens than immigrants without malaria. However, the magnitude of the response compared to semi-immune adults with malaria depended on the antigen tested. Thus, immigrants had higher IgG levels against AMA-1 and MSP-1Upon cessation of malaria exposure, IgG responses to malaria-specific antigens were maintained to a large extent, although the conservation and the magnitude of the recall response depended on the nature of the antigen. Studies on immigrant populations can shed light on the factors that determine the duration of malaria specific antibody responses and its effect on protection, with important implications for future vaccine design and public health control measures. Plasmodium falciparum infection immunity wanes rapidly, and this is in contrast with the long-term antibody-mediated immunity that follows one or few exposures to antigens from other infectious microbes [Maintenance of long-term memory responses is critical for achieving protective immunity against many pathogens. The understanding of differential immuno-reactivity to malaria and maintenance of these immune responses is fundamental for the development and design of immunogenic strategies for disease control and eradication. In malaria endemic areas, immunity is acquired gradually with age and continuous exposure, first to severe disease and ultimately to clinical malaria and high parasitemia . Neverthmicrobes .P. falciparum infections is complex, and requires the combined action of antibodies (Ab) and cell-mediated immune responses against both pre-erythrocytic and blood stages; and these two effector mechanisms are required for both anti-parasitic as well as clinical immunity [P. falciparum antigens targeted by naturally acquired IgG associated with immunity include the merozoite proteins: apical membrane antigen 1 (AMA-1), the 42-kDa fragment from the C terminus of merozoite surface protein 1 (MSP-142), and the 175 kDa erythrocyte binding antigen (EBA-175), all three involved in erythrocyte invasion [P. falciparum erythrocyte membrane protein 1 (PfEMP-1) is the major antigen of this VSA family, containing several domains that mediate cytoadherence to host cells, like the Duffy binding-like alpha (DBL-α) domain that is involved in rosetting [The control of immunity ,4. The rimmunity ,6, and dimmunity . P. falcinvasion . In addiinvasion . The P. osetting .Despite the common perception that immune memory to malaria is short-lived in the absence of exposure, most clinical evidence suggests that immunity may last for long periods of time. Immigrants maintain some immunity to clinical malaria, and have milder episodes than naïve adults with a malaria primary infection –21. AlsoHowever, data on the longevity of protective immune responses are scarce. Ab responses to malarial antigens are often thought to be short-lived –30, althP. falciparum-specific Ab responses as indicative of loss or maintenance of immunity in immigrants who have not been exposed to malaria for long periods of time. We compared IgG levels against a panel of blood stage antigens thought to be involved in malaria immunity between previously exposed immigrants, continuously exposed adults and naïve travelers, with or without a clinical malaria episode, using Luminex and flow cytometry.The aim of this study was to measure Written informed consent was obtained from participants before sample collection. Approval for the protocols was obtained from the Hospital Clínic of Barcelona Ethics Review Committee and the National Mozambican Ethics Review Committee. Parasitemic individuals were treated according to standard national guidelines at the time of the studies.P. falciparum infection (semi-immune adults), with (n=50) or without clinical malaria (n=27); and (iii) naïve adults from a non-endemic area returning from a sub-Saharan Africa malaria endemic region with a first malaria episode .Three groups of participants were recruited for this study: (i) sub-Saharan African adults originally from malaria endemic areas residing in Spain (immigrants) without clinical malaria (n=37) or with clinical malaria (n=55) upon return from travel; (ii) adults from a sub-Saharan Africa endemic area with life-long exposure to P. falciparum clinical malaria after traveling to an African country. Clinical malaria was defined as the presence of asexual P. falciparum parasites on Giemsa-stained blood smears detected by light microscopy, together with fever. P. falciparum parasitemia in blood was measured as the percentage of parasitized red blood cells. Blood samples from acute malaria episodes (day 0) and at convalescence after malaria treatment (day 7) were collected by venipuncture into vacutainers without anticoagulant for serum cryopreservation at -80° C. In addition, blood samples from 37 immigrants visiting the Tropical Medicine Units without malaria were also collected. These immigrants were healthy companions or those presenting with non-malaria diseases. Most of them had a febrile syndrome or traveler diarrhea but the following conditions were also diagnosed: giardiasis, katayama syndrome, mononucleosis syndrome EBV, pneumonia, pruritus eczema, anxiety disorder, appendicitis, dermatitis, toxic syndrome, viral infection, ketoacidosis, diabetes, headache, spontaneous abortion, bacterial lung abscess and HIV infection. Clinical and demographical data were recorded in standardized questionnaires. Data on Ab levels from travelers have been previously published [Immigrants were recruited at the Tropical Medicine Units of Hospital Clínic de Barcelona , Hospital Arnau de Vilanova  and Hospital Santa Caterina de Salt  between 2005 and 2009. Travelers were recruited at the Tropical Medicine Unit of the Hospital Clínic de Barcelona  . Fifty-fublished , but areP. falciparum clinical malaria were recruited at the Manhiça District Hospital in the context of a hospital-based study conducted at the Centro de Investigação em Saúde de Manhiça in 2006 [P. falciparum parasites on blood smears, together with fever. Blood films were Giemsa-stained, and examined using a light microscope and parasite density in blood was measured as parasites/µL. Additionally, 27 healthy semi-immune adults were recruited in a cross-sectional study in the Manhiça District between 2005 and 2006. They were adults who had not lived in a city and were not parasitemic by microscopy at the moment of collecting the blood samples. Blood samples were collected by venipuncture into heparinized tubes, and plasma samples were cryopreserved at -80° C.Semi-immune adults were from Manhiça District in Mozambique, where malaria transmission is perennial, with some seasonality and moderate intensity. Fifty semi-immune adults with  in 2006 . ClinicaPfEMP-1 involved in rosetting [42 from 3D7 and FVO strains [AMA-1 from the 3D7 strain , the recosetting  were pro strains ,51 were P. falciparum antigens were determined using Luminex xMAPTM  and the Bio-Plex 100 platform  as previously described [IgG responses to escribed ,52. A poTrav1, IETrav2, IETrav3), two pediatric isolates from Mozambican children, one with uncomplicated malaria (IECh1) and another with severe malaria (IECh2), and one isolate from a pregnant Mozambican woman (IEWoman), each of whom had the O blood group, were tested for recognition by IgG using flow cytometry as previously described [Two laboratory clones , three isolates collected from travelers to endemic regions .Recognition of recombinant proteins by Ab was considered positive if AU values were above the mean of the negative controls plus 2 standard deviations. Threshold values for seroprevalence were: 238.02 AU for AMA-1 3D7; 1134.73 AU for AMA-1 FVO; 921.18 AU for MSP-1P=0.001). Visiting countries were very heterogeneous among immigrants and travelers. Immigrants were those returning from visiting their countries of origin. Travelers came from Burkina Faso & Mali & Senegal , Burkina Faso , Burkina Faso & Mali & Ghana & Togo , Ivory Coast , Guinea-Conakry , Equatorial Guinea , Gambia & Senegal , Madagascar , Mali , Mozambique , Mozambique & South Africa , Senegal  and Sierra Leone & Senegal . Immigrants with malaria had lived a median of 7 years in Spain, whereas immigrants without malaria had lived a median of 5 years  in immigrants with malaria or without malaria. Similarly, no significant differences were found in IgG levels or seroprevalences between individuals that had migrated from their malaria endemic country of origin 5 or fewer years ago, and those that had migrated more than 5 years ago, without malaria  or with In this study we found that after long periods without continuous malaria exposure, immigrants from endemic areas without malaria still presented seropositivities of 32% to 98% for erythrocytic antigens considered as leading vaccine candidates, and immigrants with clinical malaria had similar seroprevalences than semi-immune adults also with a clinical episode However, immigrants without malaria had lower levels of IgGs to all antigens tested compared to healthy adults with continuous life-long exposure to malaria. Although this may suggest that there is some loss of immunity, in this cohort we did not observe an association between time since migration and Ab levels, which may indicate that IgG are longer-lived than usually perceived, at least in adults. In addition, we cannot discard that the healthy adults recruited in Mozambique may have been recently exposed.P. falciparum antigens has been reported after re-exposure to malaria following prolonged periods of either sustained control or low transmission, in both children and adults [Immigrants with clinical malaria had higher IgG levels compared to immigrants without malaria and compared to naïve adults with a primary infection. This may indicate that even at day of presentation to the clinics with malaria, there is boosting of Ab against a wide repertoire of antigens. Rapid boosting of Ab responses to various d adults ,40, sugg42, which are relatively polymorphic/dimorphic [42, but more consistently associated with protection from clinical malaria [PfEMP-1 domain, which is a highly diverse protein [PfEMP-1 domain, probably due to its function in mediating rosetting, and Ab against it have been associated with protection against malaria severity [P. falciparum exposure, the magnitude of Ab recall responses upon reinfection depend on the nature of the antigen, as does the pattern of acquisition of such responses upon initial parasite exposure. Nevertheless, genetic and/or environmental differences between migrants who were originally from very diverse countries, and semi-immune adults who were from a unique African endemic area, could also account for some of the differences observed between these groups.Interestingly, the magnitude of Ab responses in immigrants with clinical malaria compared to Mozambican semi-immune adults with clinical malaria varied according to the type of antigen. Three different response patterns could be identified. First, immigrants had higher levels of IgG against the merozoite antigens AMA-1 and MSP-1imorphic ,53 and himorphic –56. In c malaria ,54,55 in protein . In factseverity . Finallyseverity . Therefoseverity ,52,58 suPfEMP-1 molecules [The finding that clinical malaria in immigrants induced higher Ab levels for the more immunogenic antigens may be attributable to a B cell response inclined toward merozoite antigens instead of a broad B cell response against a wider repertoire of different antigens. In contrast, the semi-immune group had greater Ab reactivity against multiple variants of IE antigens, indicating that the response under continuous exposure may be directed toward control of parasite variants . Immunitolecules ,60. The In contrast, the maintenance of Ab responses against DBL-α and EBA-175, which have been shown to be important in protection from clinical and severe malaria, could represent evidence that immunity to the most severe manifestations of clinical malaria is largely maintained, as clinical data appear to show –20. DataP. falciparum-specific Ab memory responses after long periods without exposure to malaria, the effect of these responses on immunity is not clear. We do not know if these Ab responses would lead to protection from infection or clinical/severe malaria upon reinfection. However, immigrants returning with clinical malaria have not been protected from infection or clinical malaria in this occasion, but this does not imply per se that they have no immunity to malaria. It could be that the progression of the disease was different than the naïve or the semi-immune adults. Unfortunately, we do not have data on the progression of the disease. In addition, we cannot conclude that they have lost immunity since even continuously exposed adults may have clinical malaria, which is what we observed also with the group of semi-immune adults in Mozambique. We are probably facing two different layers of immunity, in which the maintenance of basal levels of Ab is key in the initial control of a reinfection, whereas the capacity to generate effector responses (new Ab and MBC) may lead to prevention of malaria pathogenicity and severity.Although our results show that there are P. falciparum Ab and MBC have been shown to correlate when there is recent exposure, but correlation does not seem to persist after long periods of non-exposure [Previous findings on long-lived malaria-specific MBC support maintenance of memory responses. Malaria-specific MBC were detected in adults from a low endemicity setting in Thailand and found to persist for more than 7 years without ongoing exposure . In The exposure ,39,42,45P. falciparum may be reduced [While Ab responses to erythrocytic antigens seem to be maintained to a large extent, cytokine profiling in these same individuals showed that, upon loss of exposure, control of pro-inflammatory response and tolerance to  reduced . No corr reduced  may be dThe results of this work highlight the usefulness of migrant populations’ studies to understand the maintenance of immunity to malaria and to disentangle determinants and mediators, allowing the study of memory immune responses without the interference of natural malaria exposure. Nevertheless, several limitations were faced. We could not control for the immune status of the migrants when arriving to the non-endemic area, or for the number of returns to their country of origin with possible malaria exposure and boost of the immune responses. However, none of them had a malaria episode after their migration and the exposure may have been minimal due to the few times they returned and the nature of these short trips. Longitudinal migrant cohort studies would resolve the limitations of the present study and would allow for more precise assessment of long-term immunological maintenance. Further research into the factors that determine the duration of specific Ab responses and the underlying immune mechanisms will be important for vaccine design to induce levels of immunity that are more protective and sustained than those attained by current vaccine candidates . Public Figure S1Luminex IgG curves for each tested antigen made with a pool of plasma samples from hyper-immune Mozambican adult volunteers. Data is representative from one experiment in duplicates.(DOCX)Click here for additional data file.Figure S2Representative flow cytometry data of antibodies to IEs surface antigens assay. Panels A, B, and C show the dot plots for the lab parasite R29 and panels D, F and G the dot plots for one field isolate . Samples tested were the pool of plasma samples from hyper-immune Mozambican adult volunteers , the pool from non-exposed European adults  and plasma from one migrant patient .(DOCX)Click here for additional data file.Table S1Plasma IgG levels and seroprevalence in immigrants without malaria who have been ≤ 5 years or > 5 years in a non-endemic area.(DOCX)Click here for additional data file.Table S2Plasma IgG levels and seroprevalence in immigrants with a clinical a malaria episode who have been ≤ 5 years or > 5 years in a non-endemic area.(DOCX)Click here for additional data file.