The criteria for defining severe malaria have evolved over the last 20 years. We aimed to assess the strength of association of death with features currently characterizing severe malaria through a systematic review and meta-analysis.Electronic databases  were searched to identify publications including African children with severe malaria. PRISMA guidelines were followed. Selection was based on design , setting (Africa), participants , outcome , and prognostic indicators . Quality assessment was performed following the criteria of the 2011 Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2). Odds ratios (ORs) were calculated for each study and prognostic indicator, and, when a test was assessed in at least two studies, pooled estimates of ORs were computed using fixed- or random-effects meta-analysis.A total of 601 articles were identified and screened and 30 publications were retained. Features with the highest pooled ORs were renal failure , coma score , hypoglycemia , shock , and deep breathing . Only half of the criteria had an OR > 2. Features with the lowest pooled ORs were impaired consciousness , severe anemia , and prostration .The findings of this meta-analysis show that the strength of association between the criteria defining severe malaria and death is quite variable for each clinical and/or laboratory feature (OR ranging from 0.58 to 5.96). This ranking allowed the identification of features weakly associated with death, such as impaired consciousness and prostration, which could assist to improve case definition, and thus optimize antimalarial treatment.The online version of this article (doi:10.1186/s12916-017-0906-5) contains supplementary material, which is available to authorized users. Severe malaria accounted for approximately 2 million out of 207 million estimated malaria cases in 2012 [P. falciparum parasitemia is detected in the peripheral blood smear or confirmed by a rapid diagnostic test, there is no other cause for its symptoms, and at least one of impaired consciousness, respiratory distress, multiple convulsions, prostration, shock, pulmonary edema, abnormal bleeding, jaundice, severe anemia, hypoglycemia, acidosis, hyperlactatemia, renal impairment, or hyperparasitemia is present. These criteria reflect the definition of severe malaria established by the World Health Organization (WHO) in 2000, according to which any child with positive blood parasitemia and at least one of abovementioned criteria is qualified to receive parenteral treatment [A child is diagnosed with severe malaria when asexual reatment .P. falciparum detected on blood smear and classified as “being severely ill enough to be hospitalized”, without further specifications [In recent years, a decrease in the case fatality rate of malaria has been observed . The reaications .In this context, we conducted a systematic review and meta-analysis to better understand the prognostic value of clinical and laboratory findings used to diagnose severe malaria in African children. This assessment was aimed at refining the commonly employed definition of severe malaria to then explore the possibility to define ‘moderately severe malaria’ cases that could benefit from much more accessible oral treatment.We performed a systematic literature search using Medline, Embase, Cochrane Database of Systematic Reviews, and Thomson Reuters Web of Knowledge. Study selection followed the Preferred Reporting Items for Systemic Reviews and Meta-Analyses (PRISMA) guidelines . The firStudies reporting clinical and laboratory variables, including at least 100 children aged < 15 years who were diagnosed with severe malaria according to the WHO definitions, and which allowed reconstructing of two-by-two tables made up of outcome  and presence/absence of prognostic indicator, were included in this review. Controlled trials, non-controlled trials, cohort studies, case control studies and case series, both prospective and retrospective, were considered. When necessary, authors were contacted to obtain data to construct two-by-two tables. Two independent reviewers (BG and JD) conducted this search. Two –15 consiFinally, in view of the size of the comprised population, we also considered data from the SMAC studies  in our sQuality of selected studies and their risk of biases were assessed by applying the 2011 revised version of the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2) tool . When thData on clinical features among children who survived or died were extracted by one reviewer (PS) using a standardized data extraction form and checked by the second (JD), as well as on random basis by the third (BG) reviewer. Information on characteristics , study population , and prognostic indicators was gathered. Any identified errors were re-examined and corrected accordingly.index test  and death , was constructed for each prognostic indicator. Odds ratios (ORs) were calculated to measure the association of each prognostic indicator with death. When a prognostic indicator was assessed in at least two studies, pooled estimates of ORs were calculated. A random effects meta-analysis was performed in the case of a significant heterogeneity among studies (P < 0.05). Otherwise, the fixed-effect approach was preferred. Metan command in STATA version 12 was used to perform these meta-analyses [A two-by-two table including crossing variables, analyses . ResultsA total of 601 studies were identified and screened in the systematic database search. Through the selection process presented in the flow diagram . Out of 36 identified prognostic indicators, 18 corresponded with the clinical criteria of severe malaria established by the WHO. Two forest plots displaying pooled estimates of ORs with 95% confidence intervals (CI) calculated for 17 and 18 prognostic indicators included in the WHO definition of severe malaria are captured in Figs. Prognostic indicators with the strongest association with death included renal failure , coma , hypoglycemia , shock , and deep breathing . These five indicators also had the largest CI boundaries. Respiratory distress, while having a lower OR than the five indicators mentioned above, presented a narrower CI and lower CI boundaries in line with five top indicators . Moreover, the results were also consistent upon introduction of the SMAC study, with each association being slightly larger than without the SMAC, while the association with death of the top indicators was more homogeneous for renal failure , coma , deep breathing 4.89 (95% CI 3.28–7.29), hypoglycemia , and chest indrawing . The latter entered the top five indicators (in place of shock) and also presented the lower CI boundary (>4).Two or more convulsions  were also associated with poor outcome. However, further neurological signs, such as prostration  and impaired consciousness  were not associated with death. These results are comparable to those after inclusion of the SMAC study, namely convulsions  and prostration . Neither severe anemia, with and without the SMAC studies  nor hyperpyrexia  were associated with death.The results of the meta-analysis show that there is a large variation in the strength of the association between the different WHO-defined criteria of severe malaria and death. Renal failure, coma, hypoglycemia, shock, and respiratory distress represent those with the highest prognostic value. These manifestations were also those with the highest prognostic value for death in the original paper by Marsh , which wThe current systematic review recognizes coma  ≤ 2) and deep breathing as robust prognostic factors of pediatric life-threatening malaria that can simply be determined and recorded by skilled observers in all types of settings. Deep breathing, as a crucial respiratory sign of severe malaria, is commonly a compensatory manifestation of underlying metabolic acidosis  and is mAlthough there is no definite consensus regarding the strongest predictors of death within the WHO clinical definition of severe malaria, the WHO distinguished three groups  classingP. falciparum in peripheral blood and at least one criterion of severity [The present attempt to rank clinical features according to their prognostic values was performed to potentially better distinguish children that should definitely be receiving parenteral treatment versus those that could be considered for prompt oral treatment with artemisinin-based combinations. At present, the WHO recommends injectable artesunate for all children with asexual forms of severity . In the severity  suggesteTo our knowledge, this is the first systematic review and meta-analysis of predictors of death drawn from all relevant studies of African children with strictly defined severe malaria. Methodological quality was assessed by using a priori adjusted and defined rules of the latest version of QUADAS-2 tool, which allowed better evaluation of risk of biases in several domains. In addition, this review assessed the disease severity criteria used in the SMAC studies . Indeed,The main limitation of our analysis comes from the methodological or reporting weaknesses of some studies, of which the most important one is the lack of reproducibility of reported clinical symptoms and signs. Indeed, the inter-observer (clinician) agreement on the assessment of some of the signs, such as impaired consciousness or prostration for example, can be very low. Additionally, heterogeneity between studies regarding availability of laboratory data, threshold used to define abnormality, and quality of healthcare, especially with regards to blood transfusion and management of renal failure, need to be taken into account in results interpretation. Another limitation of our review is that it did not consider combinations of clinical and laboratory features of severe malaria because of the unavailability of individual records. It has been shown that having more than one manifestation of severe malaria increases the risk of dying  and thisIn conclusion, the findings of this meta-analysis show that the strength of association between the criteria defining severe malaria and death is quite variable for each clinical and/or laboratory features (OR ranging from 0.58 to 5.96). Despite the heterogeneity of entry criteria, the individual studies provided concordant results. A ranking allowed the identification of features weakly associated with death, such as impaired consciousness and prostration, which could assist to refine case definition and thus optimize antimalarial treatment.Additional file 1: Table S1.QUADAS-2 review-specific tailored tool and instructions for quality assessment of selected studies. (DOCX 19 kb)Additional file 2:QUADAS-2 tool. Risk of bias and applicability judgments. (PDF 499 kb)


Plasmodium falciparum and P. vivax, RDTs are unable to differentiate mixed infection with uncommon parasite species, i.e., P. ovale and P. malariae [Plasmodium in peripheral blood of a young boy from a remote community health centre (CHC), Darbha, in Bastar district of Chhattisgarh state, India  of the Indian Council of Medical Research (ICMR) was established at Darbha CHC of Bastar district in 2015 to provide prompt diagnosis and treatment of malaria in difficult and conflict-affected areas, as the villages in Darbha CHC are inaccessible and this region has been under continuous attack by the Maoists. There is no public transport available in the villages because of thick forest. People visit the hospital when they come to the market to purchase their daily needs.Plasmodium species identification using standard protocol [Plasmodium, with restriction sites for enzymes XhoI and BamHI. The full length of 18S rRNA gene was amplified with Phusion High Fidelity DNA Polymerase  and cloned into pBluescript SK (+) vector through restriction and ligation using DH5α competent cells. Amplification of the partial 18S rRNA gene was also performed using vector-specific M13 forward primer and Plasmodium species-specific reverse primer from ligated product. Sequencing of 18S rRNA from clones and ligated product was done by Di-deoxy chain termination method using 3130xl genetic analyser . Genes specific to each species, P. falciparum dihydropteroate synthase gene (Pf-dhps), P. vivax dihydropteroate synthase gene (Pv-dhps), P. malariae merozoite surface protein 1 gene (Pm-msp1), and P. ovale reticulocyte binding protein gene (Po-rbp) were also amplified and sequenced [Symptomatic patients were screened for malaria by bivalent RDT, SD Bioline Malaria Antigen P.f./P.v. , and microscopy. A finger-prick blood sample was collected from patients after taking written informed consent. Patients having doubtful parasite species were identified by molecular methods. Genomic DNA was isolated and polymerase chain reaction (PCR) was carried out for protocol . Universequenced , 11.This study protocol for collection of blood samples from patients with malaria infection was approved by the Institutional Ethics Committee of NIRTH, Jabalpur (Madhya Pradesh), India.A 12-year-old boy from a very remote village attended the Darbha CHC hospital with the complaint of repeated history of fever with no other complications.3, differential leukocyte count (DLC) = neutrophils (52%), lymphocyte (45%), eosinophils (2%), monocyte (1%), and serum creatinine = 0.5 mg/dL. The bivalent RDT showed the presence of P. falciparum only. Subsequently, peripheral blood smear was found positive for P. falciparum along with some doubtful structures, which a microscopist was unable to identify. He was given oral artesunate plus sulphadoxine/pyrimethamine (AS+SP). As fever was not coming down and Hb had lowered to <8 gm/dL, the patient was referred to district hospital where he was treated with intravenous antimalarial (quinine 20 mg/kg body weight on admission followed by maintenance dose of 10 mg/kg 8 hourly) along with other supportive treatment, as per the duty physician. However, he did not take the complete treatment and left hospital against medical advice. Follow-up of the patient was not possible due to inaccessibility of the area.At the time of admission, he gave history of 4 days’ fever and his body temperature was recorded as 100.8°F. His pulse rate was 101/min and his respiratory rate was 28/m. The laboratory findings of blood test at the time of admission were Hb≤10 gm/dL, blood glucose = 88 mg/dL, total leukocyte count (TLC) = 4,000/mmP. vivax and P. falciparum were highest (19%), followed by P. falciparum, P. vivax, and P. malariae (2.5%); P. falciparum and P. malariae (1.3%); and only 1 case with all 4 species (P. falciparum causes the most severe symptoms, i.e., severe anaemia, cerebral malaria, multiorgan failure, and death [P. vivax and P. ovale, although responsible for mild infection, may persist within the liver as hypnozoites, causing relapses even after treatment with blood schizonticides [P. malariae is also mild, may persist in the human population at very low density, and may cause renal failure [P. falciparum and P. vivax affected the clinical outcome in patients [P. malariae and P. ovale are largely underestimated by microscopy [In this study, 23% mixed infections were found with 2 or more species out of 160 cases of doubtful identification . Molecul species . All thend death . P. vivanticides . P. mala failure . The ava failure , Sri Lan failure , and Van failure  revealed highest %, followpatients . It is wcroscopy . The low highest %, followcroscopy . This stPlasmodium species was found, which is very rare, and this is the first report of such a case in India.A case of mixed infection of 4 Microscopy is the gold standard for malaria diagnosis, but in case of mixed infection, 1 or more species may be missed, especially in case of low parasitemia.The study showed high proportion of mixed infection by molecular methods.High proportion of mixed infections signifies the need of adequate training of health staff involved in malaria diagnosis, which is prerequisite for malaria elimination.


Plasmodium falciparum is a predominant malaria species that infects humans in the African continent. A recent WHO report estimated 95% and 5% of P. falciparum and P. vivax malaria cases, respectively, in Sudan. However many laboratory reports from different areas in Sudan indicated otherwise. In order to verify, we selected four hundred suspected malaria cases from Aljabalain area located in the White Nile state, central Sudan, and diagnosed them with quality insured microscopy and species-specific nested PCR. Our results indicated that the proportion of P. vivax infections among suspected malaria cases was high. We found that on average 20% and 36.5% of malaria infections in both study areas were caused by P. vivax using both microscopy and PCR, respectively. This change in pattern is likely due to the recent demographic changes and high rate of immigration from neighbouring countries in the recent years. This is the first extensive clinical study of its kind that shows rising trend in P. vivax malaria cases in White Nile area, Sudan. Plasmodium parasites [Malaria is an infectious disease of humans and other animals caused byarasites . Accordiarasites . Malariaarasites . P. falciparum, while P. vivax, P. ovale, P. malariae, and P. knowlesi cause generally milder form of malaria which other than P. knowlesi is rarely fatal [Five species of plasmodia can infect humans. The vast majority of deaths in Sub-Saharan regions are caused byly fatal , 3.P. vivax is responsible for most malaria cases in Asia and Latin America but it is almost absent from most of central Africa due to the absence of Duffy antigen, the receptor which P. vivax uses to invade human erythrocytes [ P. vivax represents around 10% to 40% of malaria cases but <1% of cases in western and central Africa [hrocytes . In eastl Africa . P. falciparum. P. vivax is relatively rare; 95% of cases are caused by P. falciparum and the other 5% are caused by P. vivax [ P. vivax since it is the second most important malaria parasite species in Sudan). The objective of this study was to document the suggested rise in the proportion of P. vivax infections among suspected malaria cases in White Nile state in Sudan.In Sudan until recently the majority of malaria cases were caused byP. vivax . HoweverThe study was approved by the ethical committee of the Institute of endemic diseases, University of Khartoum. Informed consent was obtained from each patient before participation in the study.2, with population of 1.675 million. The study was conducted in Aljabalain area, 80 km south of Rabak town, the capital of White Nile state. The area is considered mesoendemic for malaria; transmission follows mainly the rainy season (July to October). Four hundred suspected malaria cases based on patient's symptoms  and signs  were chosen randomly from Aljabalain hospital and Aljabalain military hospital aged >1 year old regardless of gender. Samples were collected during the rainy season (July–September) in 2012. All suspected malaria cases were treated with a standard regimen (artemisinin based combination therapy) by the local malaria control program according to the Sudanese Malaria Treatment Guidelines in Sudan National Malaria Control Program (NMCP), and the National Protocol for Treatment of Malaria .This study was a cross-sectional study carried out in the White Nile area which is one of the central Sudan states,  P. falciparum and P. vivax. The PCR was performed at the Institute of Endemic Diseases, University of Khartoum, with quality control in place (both positive and negative control were used). Parasite genomic DNA was extracted from whole blood samples using Chelex method. A fragment of the plasmodial 18S rRNA gene was amplified by PCR and species identification was performed with species-specific oligoprobes using the following primers: for P. falciparum, rPLU5: 5′ CTTGTTGTTGCCTTAAACTTC-3′, rPLU6: 5′-TTAAAATTGTTGCATTAAAACG-3′; for P. vivax, rVIV1: 5′-CGCTTCTAGCTTAACCACATAACTGATAC-3′, rVIV2: 5′-ACTTCCAAGCCGAAG CAAAGA AAG TCC TTA-3′, as described previously [Two and a half mL of venous blood was obtained from each patient. Malaria was diagnosed using blood film microscopy and confirmed with PCR. Both thick and thin blood films were used, fields were read at least twice, and the procedure was followed according to quality control guidelines of WHO. PCR was done for botheviously .Data were analyzed using SPSS  version twentieth software.μL of blood), In our study, both males and females are affected by malaria; however more females were represented in Aljabalain hospital (64.4%) and more males were represented from Aljabalain military hospital (64%). The average haemoglobin level in all patients from the two study sites was 11.9 g/dL. The prevalence of anaemia among malaria patients was high, more than two-thirds in both study sites. The majority of malaria cases (more than 90%) had low parasite level  and 50 (25%) of malaria infections in Aljabalain hospital and Aljabalain military hospital were caused by P. vivax. The results were even higher with PCR  were detected in 2.25% of samples in average from both study areas, In both study sites, the proportion ofwith PCR ; 66  among malaria cases was found [This study was carried out in an area characterized by seasonal and unstable malaria transmission. The most remarkable result in this study was the unexpected high proportion about 40% by PCR) of0% by PCRas found . P. vivax is parasite's development of alternative mechanisms to invade human erythrocytes other than the Duffy antigen. This is a plausible explanation since P. vivax infection of Duffy negative genotype was reported previously in many African countries [ P. vivax transmission in Sudan. And it has important health policy implications since P. vivax infection requires eradication of liver stages with primaquine due to presence of dormant hypnozoites within hepatocytes [ P. vivax infection is becoming more severe especially in children, and further studies are required to understand the exact causes of this pattern [Another suggested explanation for the emergence ofountries –16. Thisatocytes , 18. Rec pattern .The sampled population (four hundred cases) of this study was selected based on their highly suspected symptoms of malaria and positive blood film microscopy done in hospitals' laboratories. Blood films were later proved only 50% positive in more quality insured settings. This obvious result of malaria overdiagnosis by a factor of two is probably due to lack of training in general hospitals, low quality control microscopy, and high work load. The same result was previously found in Tanzania where malaria was overdiagnosed by a factor of five , 21. Plasmodium vivax diagnosis, while by PCR it was 38.7%. This may be due to difficulty in differentiation between species based on a single ring form especially for untrained personnel. In this study more than 90% of slides had low parasite density (≤500 parasite/μL); this also makes differentiation between plasmodia species very difficult.PCR diagnosis for malaria is accurate especially for differentiating between plasmodia species, but it is expensive and needs well-trained personnel. In this study quality control microscopy allowed for 20% of P. vivax infections in White Nile area, central Sudan. This result has important implications for the malaria control and necessitates modification of current guidelines for the treatment of malaria in Sudan.Our study confirmed the observed high percentage of


Plasmodium falciparum malaria in HbAS and HbAA genotypes. Plasma concentrations of haem, HO‐1 and cytokines were quantified in venous blood obtained from children (9 months–5 years of age) during malaria infection, and at convalescence (baseline levels). Parasitaemia was determined during malaria infection. In patients with the HbAA genotype, there was a significant elevation in the plasma concentration of haem (P = 0.002), and a consequent increased induction of HO‐1 (P < 0.001) during falciparum malaria compared with levels at convalescence. Contrary to HbAA, plasma concentration of haem did not change in the HbAS genotypical group (P = 0·110), and the induction of HO‐1 was reduced during malaria compared with levels at convalescence (P = 0·006). Higher plasma levels of haem were observed in HbAS compared with HbAA at convalescence (P = 0·010), but this difference did not affect the levels of HO‐1 within each genotype (P = 0·450). Relatively milder proinflammatory responses were observed in HbAS children during malaria infection compared to HbAA children. Our findings suggest that a mechanism of reduced susceptibility to severe malaria pathologies by the HbAS genotype may involve the control of haem, leading to controlled levels of HO‐1 and milder proinflammatory responses during acute malaria.The controlled induction of haemoxygenase‐1 (HO‐1), an enzyme that catabolizes haem, has been shown to reduce haem, preventing pathologies associated with haem toxicity. The hemoglobin genotype HbAS confers reduced susceptibility to severe complications of malaria by a mechanism that is not well understood. Using a longitudinal approach, we investigated the effect of baseline concentrations of HO‐1 on the accumulation of haem during acute  The clinical outcome of most infectious diseases, including malaria, is mediated by complex host and pathogen factors Plasmodium parasites have long co‐existed, and co‐evolved, with the human host. This relationship has exerted extraordinary evolutionary pressure on the human genome. Consequently, humans have selected multiple genetic polymorphisms that provide intrinsic protection against severe malaria complications The Associations between the HbAS genotype and protection against severe forms of malaria disease have been established P. falciparum invasion, replication and schizont rupture leads to haemolysis and the release of haem. Thus, the accumulation of haem during malaria is expected to be involved in severe malaria complications. Indeed, reports have implicated free haem in the pathogenesis of cerebral and non‐cerebral complications of malaria in well‐defined murine models Free haem has been shown to aggravate oxidative stress; thus, inducing an excessive proinflammatory state which has been associated with several pathologies, such as acute chest syndrome and lung inflammation P. berghei ANKA‐infected mice with the HbS allele P. berghei malaria is quite different from that of P. falciparum. Therefore, it is of interest to know whether this interesting phenomenon observed in murine malaria is relevant and observable in human malaria.Recently, the induction of haemoxygenase‐1 (HO‐1) was shown to prevent the onset of experimental cerebral malaria and mortality in Based on the available evidence, we investigated the effect of the levels of haem observed in children with the HbAS and HbAA genotype on the plasma concentration of HO‐1, and the association between plasma concentration of HO‐1 and the availability of haem during malaria infection in humans. We also investigated how levels of free haem relate to the inflammatory response in the context of malaria infection.falciparum malaria than the HbAA genotype. The controlled level of haem during acute malaria infection was associated with a milder proinflammatory response in children with the HbAS genotype compared to the HbAA genotype. However, HO‐1 did not appear to be associated with reducing haem accumulation.We present data that suggest that the HbAS genotype is associated with a better control of haem accumulation during The study was conducted at Oni Memorial Hospital, a child out‐patient clinic in Ibadan, South‐Western Nigeria. The area is a holo‐endemic region characterized by year‐round malaria transmission, with seasonal peaks between June–July and November–December n = 62, HbAA = 40, HbAS = 22) . The participants comprised 140 unrelated children (between the ages of 9 and 60 months) referred for malaria tests by the attending physician. Children who tested positive to malaria rapid diagnostic tests and were confirmed by microscopy were recruited for further laboratory analyses  to detect the best fields with even distribution of white blood cells (WBC) (10–20 WBC/field) Parasitaemia was confirmed and estimated by the microscopic examination of Giemsa‐stained thick blood films for erythrocytic stages of Laboratory characterization of the haemoglobin (Hb) types was carried out by cellulose acetate electrophoresis at pH 8·9, as described by Chanarin Total haem (protein‐bound and free haem) in plasma was quantified by a chromogenic assay, as described previously Plasma concentrations of cytokines were measured in a multiplex assay using the highly sensitive xMAP technology , which allows the quantification of several biological analytes in a 96‐well format. Using this method, six proinflammatory and four anti‐inflammatory cytokines were measured in duplicate wells for each sample. The proinflammatory cytokines were tumour necrosis factor (TNF)‐α, interferon (IFN)‐γ, interleukin (IL)−1β, IL‐2, IL‐6 and IL‐12 (p70), while IL‐4, IL‐7, IL‐10 and IL‐13 were the anti‐inflammatory cytokines quantified. The levels of each cytokine were recorded after background subtraction.U‐test. Differences between plasma levels of cytokines, HO‐1 and haem were compared within and between genotypes using either two‐sided Student's t‐test or Mann–Whitney U‐test. For association analysis, Pearson's correlation was used to investigate the relationship between parasite density and plasma concentration of haem within each genotype.Statistical analyses were performed using GraphPrism version 6.01 . Parasite densities were compared between genotypes by Mann–Whitney P = 0·200) and gender (P = 0·740) when they were stratified based on haemoglobin genotypes (i.e. HbAS and HbAA). Forty children with either HbAS or HbAA genotype were found to be parasite‐free by microscopy 3 weeks later . Among children with malaria, parasite density did not differ significantly between genotypes (P = 0·467)  males and 36 (60%) females, with a mean age of 36 (range = 9–96) months. There were no significant differences in age (P = 0.026)     Fig. . In addi02) Fig. , whereas10) Fig. . Interes10) Fig. . Taken tr = 0·67, P = 0·004)           ; however, the increase was at borderline significance in the HbAA group. Plasma concentrations of IL‐10 did not differ significantly during malaria infection compared with levels at convalescence in both haemoglobin genotypes   were also quantified in the two groups at convalescence and during malaria infection. Levels observed during malaria infection were compared with levels at convalescence. Similar patterns of anti‐inflammatory response were observed within each genotype. For IL‐4 and IL‐7, plasma concentrations increased significantly during malaria infection within both genotypical groups (HbAA IL‐4 We adopted a longitudinal approach to investigate the hypothesis that chronic haemolysis in patients with the HbAS genotype leads to increased levels of haem, which consequently induces higher chronic levels of HO‐1. High chronic levels of HO‐1 would, in turn, cause the accelerated breakdown of haem released during parasite egress, thus preventing excessive inflammation. Studies in murine models have demonstrated that the accelerated breakdown of haem by the increased induction of HO‐1 prevents the onset of experimental cerebral malaria in mice Although parasitaemia did not differ between haemoglobin genotypes, plasma levels of haem were lower in patients with the HbAS genotype compared with patients with the HbAA genotype during malaria infection, suggesting that carriers of this trait might be better able to control haem during malaria infection. Further analysis revealed that parasite density correlated positively with plasma levels of haem during malaria infection in patients with the HbAA genotype alone. The correlation analysis was confirmed further from the observation of higher plasma concentrations of haem during acute malaria infection compared with levels at convalescence in the HbAA genotype, while plasma concentrations of haem did not change in patients with the HbAS genotype during acute malaria infection compared with levels at convalescence. The increase in plasma levels of haem during malaria infection in patients with the HbAA genotype can be attributed to haemolysis driven by the lysis of erythrocytes during merozoite egress Evidence from previous studies have shown that haem, the substrate of HO‐1, is also its major inducer The increased induction of HO‐1 in HbAA patients during acute malaria poses a danger. Haem‐induced HO‐1 above a certain threshold may be part of the causal pathway leading to severe diseases Free haem can activate immune cells causing immune‐modulatory imbalance, which can lead to the secretion of proinflammatory cytokines in excess Plasmodium infection. However, after Plasmodium infection, increased IFN‐γ production by various types of cells, particularly CD4+ T cells, is involved not only in protective immunity, but also in immune pathology Higher levels of proinflammatory cytokines have been shown to cause dyserythropoiesis Observations from this study provide insights into a possible mechanism of reduced susceptibility to severe complications of malaria in patients with the HbAS genotype. We showed that patients with the HbAS genotype are able to control haem during acute malaria infection. The ability to control haem effectively led to milder proinflammatory responses in patients with the HbAS genotype compared to the HbAA genotype. The effective control of haem and proinflammatory cytokines during acute malaria may explain the reduced susceptibility of children with the HbAS genotype to severe complications of malaria.T. W. A. and G. A. A. designed the study and wrote the paper. G. A. A. and O. K. A. co‐supervised the experiments and edited the manuscript.The authors declare no disclosures.


In stable transmission areas, malaria is the leading cause of anemia in children. Anemia in children is proposed as an added sensitive indicator for community changes in malaria prevalence. We report short-term temporal variations of malaria and anemia prevalence in rural Malawian children. Data from five repeated cross-sectional surveys conducted over 1 year in rural communities in Chikwawa District, Malawi, were analyzed. Different households were sampled per survey; all children, 6–59 months, in sampled household were tested for malaria parasitemia and hemoglobin levels using malaria rapid diagnostic tests (mRDT) and Hemocue 301, respectively. Malaria symptoms, recent treatment (2 weeks) for malaria, anthropometric measurements, and sociodemographic details were recorded. In total, 894 children were included from 1,377 households. The prevalences of mRDT positive and anemia (Hb < 11 g/dL) were 33.8% and 58.7%, respectively. Temporal trends in anemia and parasite prevalence varied differently. Overall, unadjusted and adjusted relative risks of anemia in mRDT-positive children were 1.31 (95% CI: 1.09–1.57) and 1.36 (1.13–1.63), respectively. Changes in anemia prevalence differed with short-term changes in malaria prevalence, although malaria is an important factor in anemia. Plasmodium falciparum malaria infection is a major contributing factor to anemia in African children, with both conditions responsible for high morbidity and mortality.1 Children below 5 years bear the highest proportion of anemia worldwide.2 Although malaria accounts for most of the anemia in African children in high transmission settings,3 malnutrition (macronutrient deficiency), human immunodeficiency virus (HIV), and helminthic infections also play roles.57 An inverse relationship between malaria parasite density and hemoglobin level exists in children—increasing parasite density leads to lower hemoglobin levels and increasing anemia prevalence.9 Higher parasite densities are seen in high- than in low-transmission settings.12 Reduction in malaria burden, owed to the scale-up of control efforts, reduces anemia prevalence in children.13 In stable malaria transmission areas, anemia prevalence/hemoglobin level has been suggested as an additional impact indicator for changes in malaria prevalence.14 Anemia prevalence is reported to be more sensitive to changes in malaria burden compared with parasite prevalence in long-term surveys.15 High-transmission settings are associated with low parasite density and high hemoglobin levels. As hemoglobin assessment for anemia is affordable and reliable in field surveys, anemia may be a suitable indicator of changes in malaria burden in community surveys.Malaria control interventions are intended to reduce malaria-related morbidity and mortality, which is challenging to define and measure in sub-Saharan Africa.16 to describe trends and associations of anemia and parasitemia in children 6- to 59-months old and discuss whether anemia prevalence can be used as an indicator of malaria prevalence.Short-term variations in community malaria and anemia prevalence during different malaria exposure seasons have not been adequately explored in repeated cross-sectional studies. These variations reflect the potential to use anemia prevalence as an indicator of malaria burden in a community. We analyzed data from one year of repeated cross-sectional household surveys16 conducted in Chikwawa district in Southern Malawi. rMIS involves repeating cross-sectional household surveys within a defined area to measure malaria indicators.17 In this case, the indicators included malaria and anemia in children aged 6–59 months.16 The objective of the primary study was to implement rMIS as a household malaria survey at subdistrict level and provide continuous and readily available estimates of malaria and anemia prevalence. rMIS was also implemented to identify hotspots. The study setting has been previously described.16 Briefly, the primary study was conducted in the perimeter of the Majete Wildlife Reserve (MWR) a rural part of Chikwawa district from April 2015 to April 2016. The rural villages in MWR perimeter are grouped into community-based organisations (CBO), averaging eight villages. Each village has a population ranging from 58 to 1,500 people. rMIS was conducted in seven CBOs, termed “focal areas A, B, and C,” with a total of 61 villages, approximately 6,600 households, and 24,500 people. The population around the reserve relies mainly on rain fed subsistence and small-scale commercial crop farming for their livelihood. Transmission of  malaria occurs throughout the year, peaking during and after the rainy season (December to May). The rainy season is associated with flooding and abundant mosquito larval habitats. The main malaria vectors are Anopheles gambiae s.s., Anopheles arabiensis, and Anopheles funestus.18The data analyzed were from a rolling malaria indicator survey (rMIS)Survey participants were children aged 6–59 months living in the MWR perimeter. Children were eligible for inclusion in the survey if 1) they slept in a household selected for rMIS the previous night; 2) they were a permanent member of the household; 3) the head of the sampled household or a legal guardian consented to their participation; and 4) they had no signs of severe illness. Children with signs of severe illness, defined as impaired consciousness, multiple convulsions, prostrations, deep breathing, circulatory collapse, clinical jaundice, and spontaneous bleeding, were urgently referred to the nearest health facility.http://www.malariasurveys.org/toolkit.cfm). The questionnaire recorded malaria symptoms (fever or history of fever in preceding 48 hours), and recent treatment (within 2 weeks) for malaria. Treatment details for fever were confirmed from the participant’s health record passport. Blood tests included a malaria parasite test using a malaria rapid diagnostic test (mRDT)  for all participants and hemoglobin level using Hemocue 301® . SD Bioline malaria Ag Pf meets the World Health Organisation (WHO) procurement criteria; it has 95% and 99% panel detection scores at low (200 parasites/μL) and high  densities, respectively, in clinical samples.19A nurse and two to four research assistants interviewed the guardians of invited children using a tablet-based questionnaire; data were collected and managed using openHDS. The questionnaire and blood testing  were adapted from standardized Malaria Indicator Survey (MIS) . Fever was temperature 37.5°C and above.20 over two consecutive nights per house. The Suna trap was set indoors one night and outdoors the other night, with the order determined by a coin flip. All Anopheles mosquitoes were separated by sex and identified morphologically according to Gillies and Coetzee.21Adult mosquitoes were sampled using a Suna trap® weather stations  in focal area B; temperature was summarized as the daily mean and rainfall as the daily total.Hourly temperature and rainfall were recorded using a HOBOA sample size of 300 households per round  was initially calculated based on a planned 12-sampling rounds within one year to exhaust all households in an entire focal area. The sample size was later adjusted, after considering logistical capacity, to 270 households  every 3 months. Adult mosquitos were collected in 3/4 of the rMIS households (67 households); this proportion was based on the logistical capacity of the research team to efficiently set the traps in the evening and collect the mosquitoes in the morning on two consecutive days.22 Briefly, based on the household locations, we used simple inhibitory sampling, a randomized probability sampling with spatial regularity constraint technique, in the first two rounds. Three subsequent samples were based on an adaptive geostatistical design (AGD) sampling.22 AGD sampling surveys used prediction results from preceding rMIS rounds to sample new locations of high estimate uncertainty for the subsequent rounds. The estimate used was malaria prevalence. The proportion of households for adult mosquito sampling was selected from rMIS households using random sampling.A household mapping of the study site was conducted between August and November 2014. The sampling process for households has been described elsewhere.22 Each household that meets the AGD constraints is equally likely to be sampled, hence, a nonzero probability of being sampled. Entomology households were also randomly sampled from the rMIS households.The initial sample in AGD is a probability-based sample, albeit restricted to induce a degree of spatial regularity into sampled locations, and increasing efficiency without a risk of subjective bias.Anemia was defined as mild, moderate, and severe using hemoglobin level cutoffs of 10–10.9 g/dL, 7.0–9.9 g/dL, and less than 7.0 g/dL, respectively. Predictor variables were mRDT result, sex, age, nutritional status, and ownership of insecticide-treated nets (ITNs). mRDT results were classified as mRDT positive or negative. mRDT-positive results were categorized “treated”  or “untreated” (not recently treated). Untreated mRDT positives were either symptomatic or asymptomatic based on the presence/absence of 1) fever and/or 2) reported malaria symptoms during the survey.23 for statistical analysis. Z-scores of weight-for-height, weight-for-age, and height-for-age were calculated using the WHO anthro R scripts (http://www.who.int/childgrowth/software/en/). Univariate and multivariate analyses of predictors for anemia and hemoglobin were also calculated. We used a modified Poisson regression model24 to determine adjusted relative risks of anemia for mRDT-positive children (because the outcome was not rare). Predictors for anemia were chosen apriori.We used R statistical package, (version 3.3.1)Ethical review and approval were provided by the College of Medicine Review and Ethics Committee in Malawi (P.09/14/1631). An informed consent was administered to the head of household or household member above 18-years old in Chichewa . Children with mRDT-positive results or anemia were managed according to national guidelines or referred to a health facility for further treatment.Within a 12-month study period, a total of five data collection rounds were completed; data collection duration ranged from 1 to 2 months. From 1,568 sampled households, 1,377 were completed, and 894 children were enrolled during the five rounds. There were 323 (36.1%) children’s households which reported owning at least one ITN . PrevaleThe total number of mRDT-positive children was 302 (33.8%), who mostly (27.9%) were untreated mRDT positive . AsymptoOverall, the mean hemoglobin level in mRDT-negative children, untreated and treated mRDT positives were 10.89 g/dL (95% CI: 10.78–11.00), 9.91 g/dL (9.68–10.13), and 9.21 g/dL (8.74–9.68), respectively. Over 80% of children aged 6- to 12-months old had any anemia. Anemia prevalence was highest in this category and gradually declined with age . Severe Anopheles mosquito density, females per house per night, was generally low; density was highest in round 2 and lowest in round 5. Overall, 247 (27.6%), 195 (21.8%) and 95 (10.5%) children were underweight, stunted, or wasted (z-score < −2 standard deviations), respectively.There were variations in temperature and rainfall during data collection, from the dry cooler season, in rounds 2 and 3, to the wet warmer season in rounds 4 and 5, respectively . The higThere is an obvious trend in asymptomatic malaria prevalence among the five rounds: an increase between round 1 and 3 followed by a steady decrease from round 3 to 5 . The varSupplemental Table 1). mRDT-positive result and stunting decreased the hemoglobin level.Overall, mRDT-positive children had a higher risk of anemia compared with mRDT negatives in both the unadjusted and adjusted . There w16By conducting epidemiological and entomological surveys every 2–3 months, we describe short-term variations in malaria parasite and anemia prevalence in one year of data collection. We used small teams of research personnel and easy-to-use diagnostic tools to conduct the survey, making it feasible for monitoring community burden as well as active case detection and treatment. Using AGD sampling, the survey efficiently measured spatio-temporal heterogeneity in the prevalence of malaria parasites.25 and, although from a cross-sectional study design, support the role of malaria infection in anemia in children.26 Malarial anemia results from loss of both infected and uninfected erythrocytes, dyserythropoiesis, and suppressed erythropoiesis.28Overall, the adjusted and unadjusted relative risks provide evidence that malaria parasitemia is associated with anemia in children. These findings are similar to studies in sub-Saharan Africa31 In the current study, data were collected frequently (every 2–3 months) over 1 year, allowing the prevalence to be measured in more seasons and at a finer scale than previously; anemia prevalence in our findings lacks an obvious trend with respect to season or parasitemia. Anemia is still an important indicator for malaria morbidity. Long-term maintenance of low malaria burden, through scaling up of interventions, is required for population effects .Trends in anemia and malaria parasite prevalence in the survey suggest that anemia prevalence in children does not indicate short-term seasonal changes in malaria parasite prevalence. In previous studies, decrease in malaria burden was associated with a reduction in anemia prevalence. These studies had longer follow up time , fewer survey points (2 transmission points), or conducted before and after an intervention.35 ITN ownership was low in the study; households may prioritize younger siblings to use better quality ITNs, leaving older ones less protected. Higher use and better quality of ITNs in younger age groups also relates to free access by the mothers during antenatal care or during the child’s vaccine; in addition, higher ITN and better quality ITN usage may result from younger children sharing sleeping space with their parents.34Older children had more malaria parasitemia compared with younger children. Studies have shown that older children are less likely to sleep in ITNs and more likely to use older, less effective, and worn out ITNs compared with pregnant women, infants, or younger children.37 In this study, anemia in children aged 6- to 11.9-months old was exceptionally high. A possible cause of anemia in younger children is iron deficiency resulting from high iron demand for rapid growth, low iron intake from low iron concentration in breast milk and weaning food, and frequent infections affecting feeding.39 There was evidence of chronic malnutrition in the study population from the high prevalence of stunting. Stunting was associated with a decrease in hemoglobin level in the multivariate analysis. The communities relied mainly on rain-fed crop farming (for their diet) which may not consistently yield adequate harvest and has low iron content. Also, children in endemic regions acquire immunity to malaria as they grow older from repeated parasite exposure; this immunity also reduces the effect of malaria parasitemia on hemoglobin level by maintaining low parasite densities.40Anemia prevalence was comparatively higher in younger than in older children, similar to other studies.43 This low hemoglobin level takes on average 10 days and supports the relatively low hemoglobin level found in children who had recently taken AMD. The use of mRDTs and information on recent treatment of malaria in the survey offers a new category for malaria exposure . This category should be considered an important determinant of hemoglobin level and anemia in children in community surveys.Hemoglobin was lower in treated mRDT-positive children compared with untreated mRDT-positive children. Studies have demonstrated an initial sharp decrease in hemoglobin during malaria treatment with artemisinin-based combination therapy before a gradual rise to normal levels after treatment completion.44 has reported higher prevalence of gametocytes in asymptomatic than in symptomatic school-aged children, highlighting the role of asymptomatic parasitemia in malaria transmission. An overall anemia prevalence of 58.7% in randomly selected households indicates that anemia is a severe public health problem in the study population.2 A further 4.1% of children overall had hemoglobin less than 7 g/dL. The prevalence of stunting in children in the study area was higher than 2015–2016 Malawi Demographic Health Survey.45 Childhood stunting irreversibly affect adult height, school performance, and adult income.46There was a high prevalence of undiagnosed and untreated anemia, parasitemia and malnutrition in the rural community. The high prevalence of asymptomatic malaria in some months of the year is especially important because this likely drives malaria transmission in subsequent months. A recent study in Tanzania47 Complex relationships of the three conditions have been previously reported with both malaria and malnutrition being risk factors for anemia: malnutrition predisposing to malaria infection and worsening its severity, and commonly coexisting risk factors for all three conditions.49 A systematic review examining the correlation between malaria and macronutrient deficiency however reported that malaria is associated more with anemia in children than macronutrient deficiency.5High burden of anemia, malaria, and malnutrition in rural communities calls for an integrated control approach.This study has limitations. mRDTs have low specificity and positive predictive value for active malaria infection in endemic settings as they detect parasite antigens which persist in the blood after treatment and clearing of parasite. By detecting cleared infections, mRDT can over-report malaria parasitemia, and therefore, caution should be taken when reporting parasite prevalence based on mRDT results. We report mRDT-positive children in separate categories: treated and untreated. Current mRDTs are incapable of determining parasite density and detecting low density parasitemia. The use of mRDTs in the survey simplified data collection and treatment in the communities. Because this was a secondary analysis, samples in each round were not powered to detect precise estimates of association between malaria and anemia.® to report anemia. Anemia mechanisms and common etiologies of anemia in children, such as soil-transmitted helminths (STH), schistosomiasis, parvovirus B19, and HIV, were not investigated in the study. Both STH and schistosomiasis have been previously reported to be high in this region although variation in prevalence with time has not been reported.52 Sickle cell and thalassemia were not tested; the objective of the primary study was to implement rMIS as an alternative household malaria survey using small district teams and field-based tests. However, two previous studies in the same region reported a sickle-Hb-gene frequency of 0.1153 and prevalence of sickle cell disease of 2%4 in 400 infants and 101 children aged 6- to 60-months old, respectively.We only used hemoglobin level measured by HemocueFrom the study findings, anemia was associated with malaria parasitemia although the short-term temporal trends between the two parameters were different. Hemoglobin level was affected by malaria treatment in the preceding 2 weeks; younger children were disproportionately affected more by anemia than older children. Rural communities have a high prevalence of undiagnosed and untreated anemia, parasitemia, and malnutrition and therefore require integrated control efforts.Supplemental Table.


Plasmodium infection develop antibodies and become semi-immune. This immunity is suppressed during pregnancy making both the pregnant woman and the foetus vulnerable to the adverse effects of malaria, particularly by Plasmodium falciparum. Intermittent preventive treatment of malaria in pregnancy (IPTp) with Sulfadoxine–pyrimethamine (SP) tablets is one of the current interventions to mitigate the effects of malaria on both the pregnant woman and the unborn child. The extent to which IPTp may interfere with the acquisition of protective immunity against pregnancy-associated malaria (PAM) is undefined in Ghana.Women exposed to P. falciparum were quantified by the enzyme-linked immunosorbent assay (ELISA).Three-hundred-and-twenty pregnant women were randomly enrolled at the antenatal clinic (ANC) in Madina, Accra. Venous blood samples were obtained at first ANC registration and at 4-week intervals (post-IPTp administration). Placental and cord blood samples were obtained at delivery and the infants were followed monthly for 6 months after birth. Anti-IgG and IgM antibodies against a crude antigen preparation and the glutamate-rich protein (GLURP) of There was a general decline in the trend of mean concentrations of all the antibodies from enrolment to delivery. The levels of antibodies in cord blood and placenta were well correlated. Children did not show clinical signs of malaria at 6 months after birth.IgG against both crude antigen and GLURP were present in placenta and cord blood and it is therefore concluded that there is a trend of declining antibody from enrolment to delivery and IPTp-SP may have reduced malaria exposure, however, this does not impact on the transfer of antibodies to the foetus in utero. The levels of maternal and cord blood antibodies at delivery showed no adverse implications on malaria among the children at 6 months. However, the quantum and quality of the antibody transferred needs further investigation to ensure that the infants are protected from severe episodes of malaria. Plasmodium parasite [Plasmodium falciparum infection increases the risk of maternal anaemia and other complications in pregnancy, often leading to abortions and stillbirths [About 50 million women who become pregnant in malaria-endemic regions each year  are infeparasite . Pregnanparasite . Malariallbirths .P. falciparum develops slowly and naturally [Plasmodium antibodies confer protective immunity [In general, immunity to aturally , 6 afteraturally –10. The immunity , which rimmunity –14. Howeimmunity .Plasmodium, pregnant women become highly susceptible to P. falciparum due to the absence of immunity to pregnancy-specific isolates that sequester in the placenta [Despite acquiring partial immunity after years of exposure to placenta , 15 resuplacenta .The parasites that infect the placenta have a special affinity to accumulate in the erythrocytes in the blood spaces of the placenta  and alsoP. falciparum infection. These problems are more common in the first and second pregnancies since the parasitaemia level generally decreases with increasing numbers of pregnancies. In Ghana, malaria during pregnancy increases maternal anaemia and low birth weight (LBW), especially in women living in rural communities [Another reason for the high susceptibility of pregnant women is attributed to various hormonal and immunological changes during pregnancy, with a leaning from cell-mediated immunity to humoral immunity. Primigravidae have higher levels of cortisol than multigravidae and therefore lose their acquired immunity more frequently and this increases their susceptibility to munities –30.falciparum malaria due to protection from high levels of maternal anti-malarial immunoglobulin G (IgG) passively transferred across the placenta [Newborn infants in endemic areas are markedly resistant to placenta . Congeniplacenta . There iplacenta . These aplacenta . Severe placenta .falciparum malaria [The use of intermittent preventive treatment of malaria in pregnancy (IPTp) has been shown to increase birth weight and reduce the incidence of LBW  as well  malaria , 48 and  malaria . HoweverThe study was conducted at the Alpha Medical Hospital in Madina, a densely populated and fast-growing, peri-urban area in the Ga-East Municipality of Accra, southern Ghana. This study was a sub-component of a larger study that looked at IPTp implementation in the Ga-East Municipality of Accra. The full details of the study setting and characteristics of the participants are described in Stephens et al. .The study participants were selected from all pregnant women making their first visit for antenatal care at the Alpha Medical Centre from July to August regardless of ethnic or socio-economic background, gestation, parity or gravid status. The inclusion criteria were pregnant women who indicated their intention to deliver at the facility and who had not previously taken IPTp for their current pregnancy.P. falciparum antibody titre. Placenta and cord blood samples were also collected at delivery. Infants born to these women were followed at monthly intervals for 6 months. The body temperature of the infants was determined at each visit. Follow-up was facilitated by the use of mobile phones.Briefly, pregnant women who met the inclusion criteria were invited to volunteer to participate in the study. Socio-demographic information was collected by questionnaire, as well as information on gravidity, parity, the use of IPTp, insecticidal-treated nets (ITNs), drug seeking behaviour, and other anti-malarial practices during pregnancy. Gestation was estimated from abdominal examination by qualified midwives and confirmed later by scan. A venous blood sample was obtained at first registration for general screening and for plasma analysis to determine pre-IPTp antibody status. Women who were eligible for IPTp administration were served by hospital staff at monthly intervals. Blood samples were then obtained 4 weeks after each IPTp administration to determine their post-IPTp 3EDTA vacutainer tubes with butterfly needles  by a qualified phlebotomist. Each tube was labelled with the subject’s unique identification number and stored in a cool box. Samples were transferred to the laboratory at the University of Ghana, Legon for further analysis. Whole blood samples were centrifuged at 2000 revolutions per minute (rpm) using a Spectrafuge  and the plasma was carefully collected and stored at −40 °C for anti-malaria antibody assays. The resulting pellet of red cells were washed twice and then cryopreserved in glycerolyte solution at −40 °C.Venous blood was collected into 5-ml KMaternal intervillous blood (IVB) was obtained at delivery by a modified placental prick method as described by Othoro et al. . An expeP. falciparum or GLURP, and a soluble crude antigen preparation from whole blood-stage parasites were determined as previously described [5 cells/well for crude antigen in plain PBS. The plates were incubated overnight at 4 °C, retrieved, washed four times with PBS/Tween 20, then incubated with 200 μl/well of blocking buffer (3% skimmed milk in 1X PBS) for 1 h at room temperature in a humidified chamber. The plates were washed as described and 100 ul of plasma pre-diluted at 1:2000 in 1% PBS/milk was added in duplicates to each well and incubated for 2 h as described. To control for inter-assay variations in the procedure, each assay plate included a pool of hyper immune plasma known to be positive for total IgG to GLURP and the schizont extract, diluted at 1:500. To the plates were then added 100 μl/well of 0.5 μg/ml peroxidase labelled goat anti-human IgG (H + L) conjugate (KPL), and peroxidase-labelled goat anti-human IgM at 0.5 μg/ml for 1 h at room temperature for the IgG and IgM respectively and incubated for 1 h. Plates were developed with 100 μl/well 3,3′ 5,5′ tetramethylbenzidine (TMB) substrate for 30 min in the dark. After incubation the reaction was stopped with 100 μl 0.2 M H2SO4 and optical density (OD) was read at 450 um with a reference wavelength of 630 um using a spectrophotometer . Optical density values were converted to concentrations using a 4-parameter curve fit using pure human IgG and IgM standards fitted on each plate.Antibodies against two antigens, a specific blood-stage antigen of escribed . BrieflyEthical clearance for the study was obtained from the Institutional Review Board (IRB) of the Noguchi Memorial Institute for Medical Research, College of Health Sciences, University of Ghana, Legon (Study Number: 004/06-07). An informed consent was obtained from each volunteer and assent was documented by signing or by thumb printing the consent form.Responses to the questionnaire were numerically coded on paper and later transferred into an electronic database. Responses and laboratory results were entered into a computer in Epi Info version 3.5. The data were then cleaned and verified. Data summaries (frequencies and distributions) were examined. Participants were grouped by gravidity and by gestation at registration. The mean values for various parameters, such as antibody levels, use of bed nets and the number of IPTp doses administered for various categories of pregnant women, were compared using standard parametric and non-parametric statistical methods using the SPSS and EPI info statistical software.For normally distributed data, comparisons between means and percentages were done by Students’ t test, ANOVA and Fisher’s exact tests as appropriate at P < 0.05 significant level. Multivariate regression was performed with the number of IPTp doses taken and antibody titre against age, parity and gestation.Some limitations are acknowledged. First, HIV status is important in antibody reduction but data were not collected and therefore were not investigated in the present study. Second, it was not possible to examine the association between malaria antibody levels and bed nets due to the low usage of ITNs. Third, although malaria transmission is seasonal with a peak during the rainy season (July to September), the very low prevalence of malaria, reported in an earlier study  among thA total of 320 pregnant women were enrolled in the study. The details of the characteristics of the study participants have been reported in . In brieThe mean concentrations of IgG against GLURP decreased from the baseline to post IPT1 and slightly increased after IPT2, dropped after IPT3 and plateaued, but imposition of trend lines showed a sharp decrease in trend from baseline to delivery. IgG antibodies against the crude malaria parasite Ag was lower than IgG against GLURP at baseline (pre-IPTp) but increased after the first dose of IPT (IPT1) and then declined until after IPT2 and then decreased at delivery   during the post-natal follow-up to justify the collection of blood samples as per the approved protocol. There are many reasons attributed to protection in childhood. These range from the biting habits of anopheline mosquitoes that have preference to for blood of adults and older children, to protection by breast milk constituents. Others are the physiologic barrier by foetal haemoglobin (HbF), resulting in the delay of parasite development and protection from severe illness . The intThe study concludes that intermittent preventive treatment reduces immunoglobulin G levels among pregnant women on IPTp due to the low prevalence of parasitaemia. The children born to the mothers were protected for 6 months and showed no clinical symptoms after follow-up and thus IPTp allowed the transfer of antibodies across the placenta. Future studies should look into other non-malarial antigens to provide better understanding of antibody transfer.


P. falciparum are susceptible to subsequent malaria infections. However, the effect of prenatal exposure to P. falciparum on parasite density during clinical malaria episodes has not been fully elucidated. This study is a component of a prospective cohort study for which initial results have been published. This component was designed to determine the effect of prenatal exposure to P. falciparum on parasite density during clinical malaria episodes in the first 24 months of life. A total of 215 infants were involved and monitored for clinical malaria episodes defined by fever (≥ 37 °C) and parasitaemia. The geometric mean parasite counts between exposed and unexposed infants were compared using independent samples t test. The effect of in utero exposure to P. falciparum on parasite density was assessed using binary logistic regression.Existing information has shown that infants who are prenatally exposed to P. falciparum was associated with hyperparasitaemia during clinical malaria episodes , while other factors were not significantly associated (P > 0.05).The geometric mean parasite count per µl of blood during clinical malaria episodes in exposed infants was 24,889  while in unexposed infants it was 14,035 , P < 0.05. Prenatal exposure to  Plasmodium falciparum malaria is a major cause of death in children in sub-Saharan Africa [P. falciparum antigens, like P. falciparum merozoite surface protein 1–19 (PfMSP1–19) and P. falciparum merozoite surface protein 2 (PfMSP2) [P. falciparum antigens affects the development of fetal immune cells and infant susceptibility to malaria infections [P. falciparum is also associated with poor pregnancy outcomes [n Africa , 2. In e(PfMSP2) –8. Prenafections –5. Furthoutcomes .During childhood, on average children experience hyperparasitaemia in severe malaria ; howeverP. falciparum on parasite density during clinical malaria episodes in infants aged zero up to 24 months.Although prenatal exposure has been demonstrated to influence susceptibility to malaria infection , the pauThe detailed description of the study area was reported earlier . BrieflyThe study design, sample population, recruitment and follow up procedures were reported earlier . BrieflyProcedures for collection of placental tissues, storage and processing, for histological examinations were reported earlier . BrieflyP. falciparum was the only parasite species identified. Parasite counts were expressed per µl of blood, assuming a leukocyte count of 8000 per µl. Levels of parasitaemia were categorized into low parasitaemia (< 1000 parasites/µl blood), intermediate parasitaemia (1000–9999 parasites/µl blood) and hyperparasitaemia .Capillary blood samples were collected at each visit for determination of parasite density and species identification. Thick and thin blood smears were prepared, the thin smears were fixed in methyl alcohol, dried, stained with 10% giemsa and examined using a light microscope at 100× objective for diagnosis and parasite quantification. Two laboratory technicians examined the slides, discrepancies in slide reading, were resolved by a third technologist and P. falciparum antigens  were described elsewhere [2SO4 per well. The optical density (OD) was measured at 450 nm. Control samples were used in each run and tested sera were defined as positive if they gave OD above the cut-off point (mean + 3SD) of negative control group born and living in Sweden. Based on the test results infants were categorized as, in utero sensitized (seropositivity for IgM) and non sensitized to P. falciparum antigens (seronegativity for IgM).Cord blood sera was collected following delivery and stored at − 20 °C at field site, then transported in ice parked cool box to Muhimbili University and transferred to – 80 °C freezers until use. Detailed procedures for determination of seropositivity and seronegativity for IgM against recombinant lsewhere . BrieflyP. falciparum and other factors  on hyperparasitaemia were assessed using binary logistic regression. The difference in the geometric mean parasite counts between exposed and unexposed was assessed using independent samples t test and the difference was judged at P < 0.05.Data were analyzed using Statistical Package for Social Sciences (SPSS), IBM version 20. In order to use parametric tests data were log transformed. Assessment of effect of prenatal exposure to Detailed description of socio-demographic characteristics of the study population was reported earlier . BrieflyP. falciparum specific antigens while 10% (n = 5), had specific IgM for PfMSP1–19 and PfMSP2 in cord blood sera and were therefore sensitized to P. falciparum antigens. All infants born to mothers without placental malaria (unexposed) had no detectable IgM against P. falciparum antigens, mean parasite count during clinical malaria episodes in exposed sensitized infants was 12,731  and in exposed-non sensitized it was 28,087  and the difference was statistically significant, P < 0.01  14,035  respectively and the difference was statistically significant (P < 0.05). Ninety percent (n = 45) of infants born to mothers with placental malaria were not sensitized to P. falciparum was significantly associated with hyperparasitaemia  during clinical malaria episodes in the first 2 years of life. Other factors; gravidity, season of birth, gender of infant, birth weight and IPT-SP were not significantly associated with hyperparasitaemia in a univariate analysis . In this study, a small proportion of 10% of the exposed infants were immunosensitized as demonstrated by IgM seropositivity, and had relatively lower parasitaemia compared to the 90% who were non sensitized. This observation indicates that early exposure to the parasite antigen may have implications on their capacity to mount appropriate immune response when naturally challenged with similar parasite antigens.The findings of this study of hyperparasitaemia in infants prenatally exposed to esponses , 15. Theantigens . AlthougP. falciparum [The vulnerability of exposed infants to high parasite density observed in this study is further corroborated by previous studies, which demonstrated that prenatal exposure to malaria may negatively affect the acquisition of antibodies against lciparum , renderilciparum , 16–20.P. falciparum predisposes the exposed infants to high parasite density during clinical malaria episodes, may be supported by observations from another study which indicated that; prenatal exposure to P. falciparum is associated with immaturity of fetal/neonatal antigen presenting cells and provide insufficient co-stimulatory signals to T cells which are important in combating parasitaemia [Furthermore, the findings in this study, that prenatal exposure to sitaemia .In the pathogenesis of malaria infection, parasite density has been demonstrated to be associated with onset of severe malaria . HoweverHyperparasitaemia leads to destruction of host erythrocytes, consumption of host glucose, stimulation of lactate formation through hypoxia and alteration of erythrocyte membrane transport systems . Thus inIn this study, gravidity, use of LLINs, birth weight, age of mother at delivery, season of birth and use of IPTp-SP were not significantly associated with hyperparasitaemia during clinical malaria episodes. However, in another study conducted in Tanzania, season and use of LLINs were significantly associated with parasite density . The obsP. falciparum antigens.Prospectively, the findings of this study further emphasize the importance of preventing malaria in women of reproductive age in order to mitigate the exposure of the fetus to P. falciparum is associated with hyperparasitaemia during clinical malaria episodes in the first 24 months of life.Prenatal exposure to Anti malaria campaigns were going on in the study area.


Plasmodium falciparum sporozoites (PfSPZ) consistently leads to parasitemia and malaria symptoms in malaria-naive adults. We used CHMI by DVI to investigate infection rates, parasite kinetics, and malaria symptoms in lifelong malaria–exposed (semi-immune) Gabonese adults with and without sickle cell trait. Eleven semi-immune Gabonese with normal hemoglobin (IA), nine with sickle cell trait (IS), and five nonimmune European controls with normal hemoglobin (NI) received 3,200 PfSPZ by DVI and were followed 28 days for parasitemia by thick blood smear (TBS) and quantitative polymerase chain reaction (qPCR) and for malaria symptoms. End points were time to parasitemia and parasitemia plus symptoms. PfSPZ Challenge was well tolerated and safe. Five of the five (100%) NI, 7/11 (64%) IA, and 5/9 (56%) IS volunteers developed parasitemia by TBS, and 5/5 (100%) NI, 9/11 (82%) IA, and 7/9 (78%) IS by qPCR, respectively. The time to parasitemia by TBS was longer in IA (geometric mean 16.9 days) and IS (19.1 days) than in NA (12.6 days) volunteers . Five of the five, 6/9, and 1/7 volunteers with parasitemia developed symptoms . Naturally adaptive immunity (NAI) to malaria significantly prolonged the time to parasitemia. Sickle cell trait seemed to prolong it further. NAI plus sickle cell trait, but not NAI alone, significantly reduced symptom rate. Twenty percent (4/20) semi-immunes demonstrated sterile protective immunity. Standardized CHMI with PfSPZ Challenge is a powerful tool for dissecting the impact of innate and naturally acquired adaptive immunity on malaria.Controlled human malaria infection (CHMI) by direct venous inoculation (DVI) with 3,200 cryopreserved  In recent decades, techniques to induce CHMI have become safer, more reproducible, and focused on assessing protective efficacy of vaccine and drug candidates. Most recently, the availability of aseptic, purified, cryopreserved Plasmodium falciparum (Pf) sporozoites (SPZ), a product called Sanaria® PfSPZ Challenge has led to further advances in standardization. After direct venous inoculation (DVI) of 3,200 SPZ with PfSPZ Challenge, all malaria-naive participants have been consistently infected.7 In contrast to previous methods, the dosage, generation, and strain of the parasite are tightly controlled and, due to cryopreservation, clinical studies have fewer requirements for infrastructure and fewer restrictions on timing and design. These advances together with present knowledge and multidimensional techniques to study immunology and metabolism led us, more than five decades following the last experiments,3 to assess whether CHMI can be used to study innate resistance and naturally adaptive immunity (NAI) to malaria. For more than 100 years,8 it has been known that NAI develops after repeated exposure to Pf.9 Nevertheless, our understanding of the underlying mechanisms is incomplete. We have also known that individuals with sickle cell trait have a reduced chance of developing severe malaria. To systematically investigate these mechanisms and identify critical targets that could be used to develop subunit vaccination approaches, a stringent and reproducible infection model with a well-defined and relevant end point is required. We assessed whether standardized CHMI by DVI of 3,200 PfSPZ in healthy, adult, lifelong malaria–exposed volunteers is a suitable model that 1) is safe and well tolerated, 2) results in parasite dynamics different from CHMI in malaria-naive adults, 3) results in parasite dynamics different between HbAA and HbAS carriers, and 4) can be used to assess acquired immunity against the pre-erythrocytic and erythrocytic stages of the Pf lifecycle.Controlled human malaria infection (CHMI) is a tool that can be used to accelerate the clinical development of antimalarial interventions.Centre de Recherches Médicales de Lambaréné (CERMEL) in Lambaréné, Gabon from July 2014 to February 2016. Lambaréné is situated in the Central African rainforest and has a perennial transmission of malaria with reduced transmission in the dry season from July to September.10 The indigenous adult population is “semi-immune” against malaria, with no cases of severe malaria and a high prevalence of asymptomatic infections.11 The prevalence of sickle cell trait is approximately 15%.12 Volunteers were healthy adults between 18 and 30 years of age, without a history of severe chronic diseases and without a history of intake of an antimalarial drug in the preceding weeks. Pregnancy and the intention to become pregnant during the study were exclusion criteria. Volunteers were screened and allocated to one of three groups: nonimmunes (NI group), who were malaria naive or minimally exposed without sickle cell trait (Europeans visiting or living in Gabon); semi-immunes (I) with lifelong malaria exposure, and with hemoglobin HbAA (IA group) or with sickle cell trait (HbAS) (IS group). Malaria naivety was defined as no history of Pf malaria and no long-term (> 5 years) residence in a malaria-endemic area. Lifelong malaria exposure was defined as long-term (> 10 years) residence in a highly malaria-endemic area. A total of 25 volunteers, 10 each for groups IA and IS and five in the NI group were planned to be recruited. These numbers would allow detection of a difference in the time to parasitemia of at least 2 days between the groups (NI<IA<IS) with more than 95% power, assuming low variation in pre-patent period in the semi-immunes. In case that CHMI with 3,200 PfSPZ would not be successful in more than 50% of IA and IS group volunteers, the dose was escalated to 12,800 PfSPZ.The study was designed to assess the effect of lifelong malaria exposure and sickle cell trait on infectivity and on time to parasitemia and to malaria-related symptoms. Primary end points resulting in treatment were clinical malaria, defined as parasitemia plus symptoms, asymptomatic asexual parasitemia ≥ 1,000 parasites/μL, or Day 28, when all nonpreviously treated study subjects were treated. PfSPZ Challenge was inoculated by DVI in a dosage of 3,200 PfSPZ. The trial was conducted at the 13 Clindamycin has a half-life of 2–4 hours14 and does not interfere with subsequent CHMI. Two days after the last dose of clindamycin, volunteers received 3,200 PfSPZ of PfSPZ Challenge by DVI (Day 0). PfSPZ Challenge consists of aseptic, purified, infectious PfSPZ, strain NF54, produced and cryopreserved according to good manufacturing practices.15 PfSPZ Challenge is reconstituted in 0.5 mL buffer and injected through DVI. Volunteers remained on site for at least 1 hour after DVI, were visited on the following day, and contacted daily thereafter. From Day 5 until treatment, volunteers were visited daily to sample blood and record symptoms. Treatment with artemether–lumefantrine was administered to nonimmunes once the thick blood smear (TBS) was positive. In semi-immunes, treatment was administered once malaria was suspected. Malaria was diagnosed after a positive TBS (at any level of parasitemia) and symptoms that could be attributed to malaria, such as headache, fever, and arthralgia were present. Volunteers with parasitemia above 1,000 parasites/μL were treated irrespective of symptoms. All volunteers without parasitemia or remaining at low density parasitemia without symptoms were treated on Day 28. Quantitative thick blood films were prepared as described,16 at least once a day. Two or more microscopists were required to observe a minimum of two unambiguous parasites to declare a slide positive. The limit of detection was adjusted to a 95% probability of detecting parasitemia of at least 10 parasites/μL. In addition, 0.5 mL blood sample was collected to measure submicroscopic parasitemia by quantitative polymerase chain reaction (qPCR) using published procedures,17 with minor modifications; blood was stored in RNAlater™ and extracted using Qiagen blood isolation kits . The lower limit of quantification of the qPCR was 20 parasites/mL; the lower limit of detection of the qPCR 5 parasites/mL. Gametocytes were detected microscopically and through qPCR of Pfs16 and Pfs25 mRNA.18 Genotyping of parasites was performed by nested polymerase chain reaction (PCR) using msp2 and glurp primers and subsequent sequencing of the msp2 gene.On screening, volunteers were evaluated for clinical and laboratory abnormalities on two occasions. In addition, hemoglobin status was assessed by gel electrophoresis. After inclusion and allocation, volunteers, including the NI group, started a regimen of 12-hourly 300 mg clindamycin for 5 days (Day 7 to Day 3), a highly efficacious and well-tolerated antimalarial regimen that targets asexual liver and blood stage parasites.19 The serum dilution at which the optical density (OD) was 1.0 was assessed and designated as ‘OD 1.0’. An individual was considered to have developed antibodies if the difference between the OD 1.0 on Day 28 and Day 0 was at least 50 and the ratio of the OD 1.0 on day 28 to day 0 was at least 3.Antibodies against the Pf circumsporozoite protein (PfCSP), exported protein-1 (PfEXP1), and merozoite protein-1 (PfMSP1) before and on Day 28 after injection of PfSPZ Challenge for CHMI were assessed by enzyme-linked immunosorbent assay as previously described.20 based on reference values from the study area. On Days 35, 84, and 168 after challenge, volunteers were seen for follow-up safety assessments, including safety laboratory tests.Symptoms and adverse events (AEs) were captured through semistructured interviews and diaries, followed by coding using MedDRA. All AEs were graded , and their relatedness to the study interventions  was assessed on a five-grade scale . Laboratory deviations were graded with a modified toxicity grading scale,Comité National d’Ethique de la Recherche) and was conducted under the US FDA Investigational New Drug application. Before enrollment, written informed consent was obtained from volunteers, and understanding of the study and procedures was assessed with a quiz. The study followed the principles of the Declaration of Helsinki in its sixth revision as well as the “International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use–Good Clinical Practice (ICH-GCP)” guidelines. Safety of participants was supervised by an independent safety review committee. The study is registered with ClinicalTrials.gov, number NCT02237586.The study received approval by the Gabonese National Ethics Committee  by independent double data entry. Data validation and statistical analysis were performed using R, version 3.3. Investigators, microscopists, and volunteers were blinded on hemoglobin status until Day 28. Blinding on malaria-exposure status was not practical because volunteers of the NI group were of European descent, and all others were Africans. Statistical analysis was performed using the log-rank test for time-to-event analysis and the Fisher’s exact test (2-tailed) for proportions, unless indicated otherwise.Supplemental Figure 1).Twenty-five healthy volunteers, 12 women and 13 men between 18 and 28 years of age, were recruited of a group of 61 volunteers who were assessed for eligibility . The fivFour volunteers had asymptomatic parasitemia before clindamycin treatment. Three had gametocytemia, two by TBS and PCR, and another one by PCR only. After treatment with clindamycin, all volunteers were free of asexual parasites 1 day before CHMI (Day C-1).P = 0.14).All (5/5) volunteers in group NI, 7/11 (64%) in group IA, and 5/9 (56%) in group IS were positive by TBS . The difP = 0.55). Of note, all four volunteers who did not develop parasitemia were female (P = 0.01).Two of the four TBS-negative volunteers in group IA and two of the four in the group IS were positive by qPCR. Thus, 9/11 volunteers in group IA and 7/9 volunteers in group IS were positive by qPCR  during tP = 0.016) and IS (P = 0.021) groups (P = 0.43) and between IA and IS groups (P = 0.47) (P = 0.64) or asexual parasite (P = 0.46) carriage at screening on time to parasitemia.The geometric mean time from injection of PfSPZ Challenge until the first positive TBS was 12.6, 16.9, and 19.1 days for the NI, IA, and IS groups, respectively . The time to parasitemia by TBS for the NI group was significantly shorter than that for the IA  groups . Times t = 0.47) . There w7 which typically have shown an approximate 10-fold exponential multiplication every 48 hours from Day 7 on. By contrast, all lifelong malaria–exposed adults controlled, at least partially, parasite multiplication, although an actual rate could not be calculated because of the irregular patterns . The pattern of parasitemia over time and the area under the curve were similar between groups IA and IS. There was a considerable variation that can be explained in part by technical problems during storage and extraction of nucleic acids. As a consequence, no parasites were detected in some samples that in reality had low parasitemia. Parasitemia associated with a spike appearing late in the follow-up was genotyped. All parasites were found to be identical to the CHMI strain.Parasite growth measured by qPCR in the NI group was consistent with previous trials in nonimmunes,P = 0.003), but there was no difference between NI and IA groups (P = 0.12) (P = 0.04).The study protocol called for treatment of all volunteers in the NI group at the first detection of parasitemia, but treatment of IA and IS volunteers was given only if they had clinical malaria or asymptomatic parasitemia ≥ 1,000 parasites/μL by TBS. One volunteer in the IS group presented with parasitemia ≥ 1,000 parasites/μL  without symptoms and was treated as per the protocol. All volunteers in the NI group, but only 6/11 volunteers in the IA group and 1/9 volunteers in the IS group, developed parasitemia and symptoms. The difference between NI and the IA and IS groups combined was significant ( = 0.12) . The difP = 0.09).In volunteers with clinical malaria, time between positive TBS and first malaria-attributed symptoms was different between malaria-naive (0 hours) and lifelong–malaria exposed adults, but it did not reach the level of statistical significance (P = 0.49) or asexual parasite (P = 0.23) carriage at screening on time to clinical malaria.There was no significant effect of sexual (P = 0.0014) of volunteers who did and did not become parasitemic by TBS developed antibodies to PfEXP1, and 9/12 and 1/8 (P = 0.02) developed antibodies to PfMSP1 , 9 (45%), and 9 (45%) of the 20 semi-immunes and 0, 1 (20%), and 2 (40%) of the five nonimmunes developed antibodies to PfCSP, PfEXP1, and PfMSP1, respectively . Not suro PfMSP1 .P = 0.046; PfEXP1: 0.96 versus 41, P = 0.003; PfMSP1: 1.3 versus 52, P = 0.04) .Semi-immune subjects who had parasitemia with symptoms had a more pronounced increase in antibodies against all three antigens from baseline to Day 28 compared with those with asymptomatic parasitemia (Wilcoxon test comparing the ratio OD 1.0 on Day 28 to before CHMI (Day 0) for symptomatic versus asymptomatic individuals: PfCSP: 1.0 versus 1.7, Injection of PfSPZ Challenge was well tolerated, and no serious AE occurred during the trial. Every volunteer had at least one AE, and a total of 470 AEs were recorded over 208 days of observation.Pretreatment with clindamycin was well tolerated with 21 mild events in 10 volunteers. During the 5 days after injection of PfSPZ Challenge, there was one related local reaction (injection site pruritus) and four systemic events . Between 6 and 28 days after injection of PfSPZ Challenge, there were 314 AEs recorded in this period, of which 224 (71%) were related to malaria. Malaria-related symptoms were more severe in nonimmunes , but no Supplement Table 1). Of these, five occurred during a malaria episode, and all normalized in the course of follow-up.Of 1,663 laboratory measurements, 130 (8%) were out of range, but none were considered as clinically significant. Seven of these were grade 3 on the toxicity scale .In this study, we observed that all malaria-experienced volunteers controlled parasitemia; some manifested sterile protection, others exponential growth but with lower parasite multiplication rates compared with malaria-naive volunteers. The ability to control parasitemia was shown most strikingly by the differences between the time to parasitemia of the five nonimmune volunteers and that of the volunteers with lifelong exposure. Furthermore, all five nonimmunes developed clinical malaria, whereas only seven of 20 malaria-exposed volunteers did  volunteers with no or minimal malaria exposure have become parasitemic.7 Before that, CHMI studies in lifelong malaria–exposed subjects were conducted using locally infected mosquitoes, preparations thereof  and blood from donors with high asexual parasitemia.9 In some cases, these methods were combined. By contrast, PfSPZ Challenge, similar to Sanaria PfSPZ Vaccine, which is the same product but radiation attenuated,15 is derived from a master cell bank and produced according to good manufacturing practice guidelines. Therefore, volunteers are infected with exactly the same material and, when given by DVI, using a tightly controlled dose, an approach that cannot be reproduced using mosquitoes. Consequently, expression patterns of immunologically variant antigens, i.e., var genes in malaria-naive volunteers, are conserved,26 a prerequisite to understand naturally acquired immunity.Two studies using PfSPZ Challenge have been performed in African adults and have been published.8 and serves as a main argument for developing asexual blood stage vaccines. The strongest evidence for naturally acquired immunity (NAI) after lifelong exposure comes from passive transfer experiments using antibody preparations from lifelong malaria–exposed individuals to treat malaria patients with no or low-level immunity,29 but there are recently minimal data on how NAI influences infection kinetics using modern clinical trial and diagnostic methodology.Resistance to severe and complicated malaria in lifelong-exposed adults has been investigated for more than 100 yearsTo our knowledge, this is the first time that naturally acquired sterile protection has been confirmed using highly sensitive diagnostic methods, and that patterns of parasite control have been characterized in semi-immune subjects. One can only speculate how the four volunteers achieved sterile protection; potential mechanisms include 1) prevention of infection of hepatocytes, 2) elimination of infected liver cells by direct T cell–mediated cytotoxicity or by immune mediators , 3) highly effective clearance of the first generation of merozoites leaving the infected hepatocyte, or a combination of these factors. In this study, antibodies to PfCSP, PfEXP1, or PfMSP1 before CHMI were not associated with development or lack of development of parasitemia. Therefore, we assume that a combination of the above-mentioned adaptive immune mechanisms is the most likely reason for the differences in time to parasitemia between nonimmunes and semi-immunes. Because no method to measure liver load in humans is available, it will be challenging to completely elucidate the mechanism of naturally acquired sterile immunity. Other insights may come from in-depth immunology analyses currently being performed with samples from this study. These will be published separately.5 but all subsequent trials with malaria-naive4 and lifelong-exposed subjects3 showed no significant effect of sickle cell trait on infection rate and early parasite kinetics. Our data are in agreement with the latter studies showing no large effect of sickle cell trait on probability of infection; however, a significant difference was observed in occurrence of disease. Only one of the nine volunteers with sickle cell trait developed clinical malaria. This is corroborated by our recent data from a vaccine trial in 34 semi-immune subjects with subsequent CHMI . Aggregating both studies showed a significant effect of HbAS in preventing symptoms with 20/40 volunteers in the IA groups combined developing parasitemia with symptoms compared with 2/14 in the IS groups (P = 0.027).More than 60 years ago, CHMI was used to study the effect of NAI and sickle cell trait on malaria. One study showed a high rate of protection in sickle cell trait carriers from Uganda compared with subjects with normal hemoglobin,12 to a nonsignificant trend of longer reinfection times in HbAS individuals30 to a strong effect of increased time to reinfection.32 Some of this discrepancy may be explained by characteristics of the sample and end points. Although CHMI with PfSPZ is the most stringent approach to address this issue, the inoculum dose may have an important effect on infection rate and is likely to be lower under natural exposure compared with CHMI. Our study showed somewhat longer times to develop parasitemia in those with sickle cell trait, suggesting that a repeat study with a larger sample size might be instructive.Apart from the well-described effect of sickle cell trait on morbidity and mortality, epidemiological data on time-to-reinfection as a measure of infectivity under natural exposure in different transmission settings are also inconsistent, ranging from no effect of hemoglobin genotype on time to reinfectionIn summary, our data show that even with small sample sizes, CHMI with PfSPZ Challenge can be used to gain insight into the impact of NAI and innate factors  on incidence of infection with Pf and symptoms associated with infection. NAI prolonged the time to parasitemia, and sickle cell trait may have prolonged it further. NAI alone did not have a significant impact on the incidence of symptoms associated with parasitemia, but the combination of NAI and sickle cell trait significantly reduced the clinical manifestations of Pf malaria.Supplemental Figure and Table.