Factors driving inter-individual differences in immune responses upon different types of prenatal malaria exposure (PME) and subsequent risk of malaria in infancy remain poorly understood. In this study, we examined the impact of four types of PME  on both spontaneous and toll-like receptors (TLRs)-mediated cytokine production in cord blood and how these innate immune responses modulate the risk of malaria during the first year of life.We conducted a birth cohort study of 313 mother-child pairs nested within the COSMIC clinical trial (NCT01941264), which was assessing malaria preventive interventions during pregnancy in Burkina Faso. Malaria infections during pregnancy and infants’ clinical malaria episodes detected during the first year of life were recorded. Supernatant concentrations of 30 cytokines, chemokines, and growth factors induced by stimulation of cord blood with agonists of TLRs 3, 7/8, and 9 were measured by quantitative suspension array technology. Crude concentrations and ratios of TLR-mediated cytokine responses relative to background control were analyzed.Spontaneous production of innate immune biomarkers was significantly reduced in cord blood of infants exposed to malaria, with variation among PME groups, as compared to those from the non-exposed control group. However, following TLR7/8 stimulation, which showed higher induction of cytokines/chemokines/growth factors than TLRs 3 and 9, cord blood cells of infants with evidence of past placental malaria were hyper-responsive in comparison to those of infants not-exposed. In addition, certain biomarkers, which levels were significantly modified depending on the PME category, were independent predictors of either malaria risk (GM-CSF TLR7/8 crude) or protection  during the first year of life.These findings indicate that past placental malaria has a profound effect on fetal immune system and that the differential alterations of innate immune responses by PME categories might drive heterogeneity between individuals to clinical malaria susceptibility during the first year of life.The online version of this article (10.1186/s12916-018-1187-3) contains supplementary material, which is available to authorized users. Plasmodium falciparum infection during infancy ). Few infants were born to mothers with acute PM at delivery (2.2% [7/313]). There was a higher but non-significant proportion of females than males among the newborns (P = 0.158).The characteristics of the participants included in this study are presented in Table 10 of ratios of stimulated and unstimulated samples for each TLR agonist , while those significantly induced in response to TLR9 included IFN-α, IL-1RA, MCP-1, and IP-10 . For TLR7/8 stimulation, all analyzed cytokines  had a significantly higher concentration than that of unstimulated samples .Overall variance of cytokine responses between subjects and stimuli is shown in Fig. P < 0.05). As expected, there was little variation in cytokine levels according to PME category following stimulations by TLR3 or TLR9, as these PRRs ligands induced very low cytokine production. The concentrations of cytokines in unstimulated samples also differed between PME categories. Overall, there was a tendency of lower cytokine levels among infants prenatally exposed to malaria (any category) than in non-exposed infants . The comparison of biomarker levels in unstimulated samples from the exposed infants did not show a significant difference between PME groups. However, the trend analysis revealed a significant trend towards decreasing production among unstimulated samples from infants born to mothers with peripheral infection to those born to mothers with PM  for some biomarkers including IL-10 (P for trend = 0.024), IL-12 (P for trend = 0.042), and GM-CSF (P for trend = 0.032).Variation in cytokine production by PME category is shown as boxplots in Additional file P < 0.05, Table P = 0.026 and P = 0.008, respectively), but lower IL-5 responses . Finally, TLR9-mediated FGF and G-CSF responses were found to be significantly higher in infants born to mothers with past PM compared to the control group , whereas IL-5 responses were significantly lower in infants born to mothers with chronic PM compared with those in the non-exposed control group (P = 0.009).Innate immune response to TLR stimulation by PME was further investigated using multivariable linear regression models. The co-factors, besides PME, affecting cytokine responses in each stimulation assessment used in subsequent models are listed in Additional file Results from the multivariable models confirmed decreased levels of cytokines in unstimulated samples from infants prenatally exposed to malaria compared to the non-exposed control group: lower cytokine responses were found in past PM exposed group , chronic PM , acute PM (IL-7 only), and for peripheral infections during pregnancy .Data on malaria incidence and prevalence among the overall birth cohort has been described elsewhere . In the Using crude concentration of cytokines, we found that higher concentrations of eotaxin (in both unstimulated and TLR7/8-stimulated samples), IL-7 (in TLR3-stimulated samples), GM-CSF (in TLR7/8-stimulated samples), and IL-1β (in TLR9-stimulated samples) in the cord blood at birth were significantly associated with an increased risk of subsequent clinical malaria episodes during the first year of life Table  and 3 inIn this study, we investigated the impact of different manifestations of malaria in pregnancy on both spontaneous and TLR-mediated cytokine production by cord blood cells at birth and we assessed whether these cytokines predicted malaria risk/protection in infancy. Overall, we found that PME has a profound effect on the fetal immune system and that the differential modulation of infants’ innate immune responses by PME could have important implications with regards to malaria susceptibility in infancy. Indeed, we observed that spontaneous cytokine, chemokine, and growth factor production were all significantly lower in samples from exposed versus non-exposed infants. However, following TLR7/8 stimulation, cord blood cells from mothers with past PM (pigment only) were hyper-responsive in comparison to those without evidence of prenatal exposure. Importantly, we identified some responses (both spontaneous and following TLR stimulation) associated with differential malaria risk in infancy.+ T regulatory, and effector cells [To our knowledge, this study reports for the first time the effect of these categories of PME on TLR-mediated innate immune responses, as previous studies have focused on the overall effect of PM and/or other types of PME on PRR-mediated cytokines responses , 30, 49.or cells –15. Inteor cells –54 and tor cells . TherefoWhile TLR7/8 stimulation induced robust cytokine responses, overall cytokine responses induced by TLR3 and TLR9 stimulations were low with limited variations between PME categories, consistent with previous investigations in African children , 55, 56 Innate immune activation plays a crucial role in host protection as well as pathogenesis during malaria infection , 60. TheNotably, we observed that some cytokines, which were associated with PME, were independent predictors of malaria risk or protection, demonstrating the clinical relevance of the modulation of infants’ innate immune responses by PME. However, few studies have investigated the predictive value of cytokines, measured at birth either in unstimulated samples , 61 or uIn this study, two main limitations should be noted. First, some of the PME groups including acute PM and non-exposed groups were small in comparison with others. Therefore, we cannot exclude an underestimation of the effect of acute PM on innate immune responses measured. However, this number reflects the prevalence of PM categories in the main COSMIC trial as most of malaria infections in the placenta were past or chronic PM (95.5%). The relatively limited number of non-exposed controls is due to the high malaria transmission in the study area and the strict definition and recruitment that we applied to this group, where pregnant women had negative RDT/LM and qPCR results at each screening and ANC visit, in addition to negative placental histology. Second, in this study, the measurement of white blood cells population and lymphocyte subsets in cord blood at delivery was not performed while there is evidence that PME can alter myeloid subsets abundance and thus influence TLR-mediated innate immune responses. Therefore, the lack of these information has eventually limited the interpretation of our data.In conclusion, despite these limitations, our findings indicate that the various PME categories have different effects on innate immune responses of the newborn at birth, which might drive variation between individuals to malaria susceptibility during the first year of life. The differential alteration of TLR-mediated immune responses by PME categories may have profound implications on immune responses to other infections as well as to vaccines formulated with TLR-based adjuvants in infants prenatally exposed to malaria.Additional file 1:Figure S1. Boxplots generated using log10 of crude concentrations (pg/mL). Figure S2. Boxplots generated using log10 of ratios (TLR/unstimulated). (PDF 287 kb)Boxplots showing variation in cytokine responses by stimulation. Additional file 2:Figure S3. Boxplots generated using log10 of unstimulated crude concentrations (pg/mL). Figure S4. Boxplots generated using log10 of TLR3/unstimulated ratio. Figure S5. Boxplots generated using log10 of TLR7/8/unstimulated ratio; Figure S6. Boxplots generated using log10 of TLR9/unstimulated ratio. Non-expo, non-exposed; expo-no-PM, exposed/no placental malaria; past PM, past placental malaria; chronic-PM, chronic placental malaria; acute PM, acute placental malaria. (PDF 350 kb)Changes in cytokine responses according to prenatal malaria exposure categories. Additional file 3:Table S1. Variables included in the linear regression models assessing the effect of prenatal malaria exposure on TLR-mediated cytokine responses at birth. (PDF 88 kb)


As TNF/IL-10 coproducers, CD16+ DCs contribute to early inflammatory processes, yet P falciparum restimulation skewed cytokine responses further towards IL-10 production.This study identified CD16 Plasmodium subverts host immune responses by several strategies including the modulation of dendritic cells (DCs).The malaria causing parasite Plasmodium falciparum skewed CD16+ DC cytokine responses towards interleukin (IL)-10 production in vitro, distinct to the cytokine profile induced by Toll-like receptor ligation. To determine CD16+ DC responsiveness in vivo, we assessed their function after induced P falciparum infection in malaria-naive volunteers.In this study, we show that + DCs underwent distinctive activation, with increased expression of maturation markers human leukocyte antigen (HLA)-DR and CD86, enhanced tumor necrosis factor (TNF) production, and coproduction of TNF/IL-10. In vitro restimulation with P falciparum further increased IL-10 production. In contrast, during naturally acquired malaria episode, CD16+ DCs showed diminished maturation, suggesting increased parasite burden and previous exposure influence DC subset function.CD16+ DCs as the only DC subset activated during primary blood-stage human Plasmodium infection. As dual cytokine producers, CD16+ DCs contribute to inflammatory as well as regulatory innate immune processes.These findings identify CD16 Plasmodium falciparum [Plasmodium falciparum is known to subvert host immune responses by a range of strategies [+ DCs and CD1c+ DCs, and a third subset CD16+ DCs, characterized by surface expression of CD16/FcγRIII [+ DCs comprising half the myeloid DC compartment [P falciparum infection is unknown. How CD16+ DCs respond to parasite antigen in vitro, after experimentally induced P falciparum infection or during naturally acquired malaria, remains to be defined.Malaria is a major global health problem, causing almost half a million deaths in 2016, the majority of which were attributable to rategies , 4. As prategies . Human p/FcγRIII . Despitepartment , reportsPlasmodium parasite can disrupt monocyte-derived DC function in vitro [P falciparum-infected red blood cells (RBCs) has been reported [+ DCs were not evaluated. In clinical malaria, myeloid DC function in general is compromised [+ DCs, yet expression of costimulatory molecule CD86 is increased compared with age-matched uninfected controls [P falciparum infection on CD16+ DCs in adults remains unclear.Seminal studies have shown the in vitro , 10. Morreported , whereaspromised , 12, 13.controls . The imp+ DCs has been ambiguous, and some degree of controversy remains around whether these cells classify as DC or nonclassic monocytes  including TLR1, TLR2, TLR4, and TLR7 [+ DC cytokine production in response to lipopolysaccharide (LPS)/interferon (IFN)-γ stimulation, with CD16+ DCs considered an inflammatory DC subset in autoimmune diseases [+ DC cytokine profile in response to parasite stimulation in vitro or ex vivo after Plasmodium infection is yet to be reported.Historically, the classification of CD16iewed in ). Initiaiewed in , subsequiewed in . Howeveriewed in  and singand TLR7 , equippidiseases  and in hdiseases . The CD1Plasmodium parasites under close clinical monitoring [Plasmodium infection. During induced malaria after P falciparum sporozoite infection, CD16+ DCs increase HLA-DR and CD86 expression [+ DCs may respond differently to very early blood-stage P falciparum infection. In this study, we describe for the first time the effect of Plasmodium parasites on human blood CD16+ DC function. We examine CD16+ DC maturation and cytokine responses during induced blood-stage P falciparum malaria (IBSM) and after in vitro stimulation. We compare these responses to CD16+ DC activation in Indonesian children and adults with acute P falciparum malaria.Induced malaria studies, in which malaria-naive volunteers are experimentally infected with nitoring , allow tpression . In contpression  and plaspression , suggestP. falciparum-infected RBCs (pRBC) as previously described [Thirty-nine volunteers, aged 19–41 years , consented to participate in a phase Ib clinical trial testing the efficacy of antimalarial drugs: ACTRN12611001203943, registered November 23, 2011; ACTRN12612000323820, registered March 21, 2012; ACTRN12612000814875, registered August 3, 2012; ACTRN12613000565741, registered May 17, 2013; ACTRN12613001040752, registered September 18, 2013; and NCT02281344, registered October 3, 2014. Blood-stage parasitemia was initiated by inoculation of 1800 escribed .+ DCs were assessed in cryopreserved peripheral blood mononuclear cells (PBMCs) collected from adults and children with uncomplicated P falciparum malaria as part of artemisinin combination therapy efficacy studies in Southern Papua, Indonesia [Phenotype and activation of CD16ndonesia . The PBM+ DCs were characterized as lineage- , HLA-DR+, CD11c+, CD123−, and CD16+. In brief, 200 µL whole blood or 3M PBMCs were stained with the following surface antibodies: CD3 (HIT3a), CD14 (HCD14), CD19 (HIB19), CD20 (2H7), CD34 (561), CD56 (HCD56), HLA-DR (L243), CD11c (B-Ly6), CD123 (6H6), CD16 (3G8), and CD86 (2331). All antibodies were purchased from BD Biosciences or BioLegend. For whole blood, RBCs were lysed with FACS lysing solution (BD Biosciences) and cells were fixed with 1% (w/v) paraformaldehyde in phosphate-buffered saline (PBS). Absolute numbers of CD16+ DCs were determined by adding automated lymphocyte and monocyte counts (109 cells/L), dividing the sum by 100, multiplying the percentage of CD16+ DCs, and multiplying the product by 1000 to give cell count/µL. All assays were performed within 2 hours after blood collection because CD16 expression was partially lost from CD16+ DCs when blood was kept overnight at ambient temperature.CD16+ DC cytokine production was assessed in 1 mL fresh whole blood unstimulated or stimulated with TLR agonists: TLR1 - Pam3CSK4 100 ng/mL; TLR2 - HKLM 108 cells/mL; TLR4 - Escherichia coli K12 LPS 200 ng/mL; or TLR7 - imiquimod 2.5 µg/mL (Sigma-Aldrich), pRBC, or uninfected RBC (uRBC) prepared as previously described [2. At 6 hours, cells were stained to identify CD16+ DCs: CD14− (M5E2), lineage −, CD1c− (L161), HLA-DR+ (L243), CD86+ (IT2.2). IgG2b isotype control was used to aid identification of CD16+ DCs. The RBCs were lysed with FACS lysing solution (BD Biosciences), washed with 2% fetal calf serum/PBS, and cells permeabilized with ×1 Perm/Wash (BD Biosciences) and stained with intracellular anti-tumor necrosis factor (TNF)-α (MAB11), interleukin (IL)-12/IL-23p40 (C11.5), IL-10 (JES3-9D7), or IgG1 isotype controls (BioLegend). To determine the stimulant-specific response, spontaneous cytokine production was subtracted from responses to pRBC, uRBC, or TLR agonists.CD16To verify CD16/FcγRIII loss during culture, we assessed DC subset phenotype in 6 unexposed controls. Fresh whole blood staining and the intracellular cytokine assay were performed using the same methods detailed above. Verification of gating strategy used the same antibodies detailed above. FACS data were acquired using a FACSCanto II (BD Biosciences) or Gallios (Beckman Coulter), and data were analyzed using Kaluza 1.3 (Beckman Coulter).http://exon.niaid.nih.gov) [P < .05.Statistical analyses used GraphPad Prism 6 (Graphpad Software Inc.) and SPICE version 5.3  . The Wil+ DCs is hampered by the observation that CD16 is lost from the cell surface during short-term culture (6 hours) [+ DCs, we determined a gating strategy using CD86, a costimulatory molecule highly expressed on CD16+ DCs when compared with other blood DC subsets  (+ DCs fell into the CD86-negative gate (+ DCs (data not shown), highlighting the importance of performing DC assays on fresh whole blood as soon as possible.Measuring cytokine production by CD166 hours)  and afte6 hours) . To circ subsets . By excl subsets , we coulmulation . CD1c+ Dreported   . Only veive gate . It is n+ DC cytokine production in response to TLR ligands, using the above gating strategy , a modest IL-12 response , and a modest IL-10 response   producing TNF. There was no detectable TLR7-triggered IL-12 or IL-10 response   and C. Iresponse . Booleanroducers .+ DCs significantly increased TNF (P = .02) and IL-10 (P = .008) production, compared with control cells stimulated with uRBC (+ DC) after pRBC stimulation significantly differed from evaluated TLR stimulations . Together, in vitro stimulation data indicate that CD16+ DC cytokine responsiveness is multipotent and stimulation specific.Next, we assessed responsiveness to pRBC. Upon pRBC stimulation, CD16ith uRBC  and B. Bith uRBC . The quaulations . InterleP falciparum on CD16+ DCs in vivo, whole blood was collected and processed from volunteers participating in an induced blood-stage P. falciparum malaria (IBSM) clinical trial. Volunteers were inoculated intravenously with ~1800 pRBC, and peripheral parasitemia was monitored by real-time polymerase chain reaction, until a predetermined threshold of ~1000 pRBC/mL was reached, at which point patients were treated with an antimalarial regimen [+ DCs spontaneously produced IL-10, IL-12, or TNF ex vivo, with TNF  dominating the response. At peak parasitemia, spontaneous TNF cytokine production increased significantly to 15% , indicating that CD16+ DCs were activated in vivo during a first P falciparum infection (P = .01) and TNF/IL-10 coproducing CD16+ DCs (P = .04) , with no significant changes in single TNF or IL-10 production  and IL-10  but not TNF production  . Next, woduction . There w to uRBC . In respoduction . Taken t+ DCs in fresh whole blood (+ DCs in the blood (P = .8)   class II (HLA-DR), costimulatory molecule CD86 (B7-2), and antibody receptor CD16 (FcγRIII). The loss of the latter has been associated with CD16+ DC activation [+ DCs with increased HLA-DR (P < .0001) and CD86 (P = .0002) expression and reduced CD16/FcγRIII expression (P = .003)  . HoweverP = .02) , reflecthe blood . CD16+ Dtivation . During  = .003) . The obsle blood .+ DCs in adults and children living in an area of perennial unstable malaria transmission, using cryopreserved PBMCs collected during an acute clinical episode of P falciparum malaria or during convalescence     . The HLActively)  and C. Ictively)  and 3B. d adults . At convP = .03) . Taken t+ DCs undergo unique and distinctive activation in response to a primary subpatent P falciparum infection, with enhanced expression of HLA-DR and CD86, reduction in CD16/FcγRIII, and increased coproduction of IL-10 and TNF. CD16+ DC activation during induced P falciparum blood-stage malaria is consistent with the activation of this subset during induced Plasmodium vivax blood-stage malaria [P falciparum sporozoite infection [Plasmodium activation of CD16+ DCs after Plasmodium infection in naive healthy adults. We have recently shown that (1) both CD1c+ DCs and plasmacytoid DCs are not activated during induced P falciparum or P vivax blood-stage malaria [+ DC subset is drastically reduced in induced P vivax blood-stage malaria [+ DCs as the only blood DC subset activated during induced human Plasmodium malaria, suggesting that CD16+ DCs may be involved in the initiation of immune responses required for parasite control during a primary infection.Our results illustrate that CD16 malaria  and indunfection , suggest malaria , 24 and  malaria . Togethe+ DC cytokine production during subpatent P falciparum infection. We show that CD16+ DCs respond to TLR1/2 and TLR4 stimulation with robust TNF production both before and during induced blood-stage malaria. Tumor necrosis factor production in response to TLR stimulation is consistent with prior reports identifying CD16+ DCs as an inflammatory DC subset [+ DCs expressing the carbohydrate modification 6-sulfo LacNac (slan) [+ DC [+CD16+ inflammatory monocytes [+ DCs also contribute to TNF production in early infection and potentially during clinical malaria.Using an indirect gating method, we characterized CD16C subset , 21, incc (slan) . Unlike c (slan)  and the c (slan) , 33, as n) [+ DC . Tumor nn) [+ DC , particuonocytes , and γδ onocytes , as the + DCs stimulated with P falciparum was proportionally greater compared with IL-10 responses induced by TLR stimulation, whereas the TNF response was comparatively modest. A recent report using isolated blood CD1c+ and CD141+ DCs showed no production of TNF, IL-10, or IL-12 by these DCs upon pRBC stimulation when compared with TLR4 stimulation [+ DCs were not included. From the data presented here, it is evident that CD16+ DCs do have the ability to respond to TLR and pRBC stimulation, yet with distinct cytokine profiles; setting them apart from classic blood DCs. In addition to increased spontaneous TNF production after P falciparum infection, CD16+ DCs became multipotent cytokine-producing cells increasing coproduction of TNF and IL-10, particularly upon Plasmodium re-exposure in vitro, which further skewed the CD16+ DC cytokine profile towards IL-10 production. Interleukin-10 producing DCs can contribute to immunoregulatory processes through the induction of highly suppressive regulatory T cells [low IL-10high type 1 regulatory (Tr1) cells [P falciparum infection, and parasite-specific IL-10 production by these cells suppresses proinflammatory cytokine production [+ DCs reported here suggests these DCs may have a role in the induction of regulatory cells in primary P falciparum infection. Such suppressive T-cell responses may blunt protective immune responses and result in higher parasite burden [+ DC IL-10 production [+ DCs contribute to both inflammatory and regulatory immune processes. Further evaluation of cytokine production by CD16+ DCs and how they impact downstream immune responses is needed.Interleukin-10 production by CD16mulation ; however T cells , 37 and 1) cells . We receoduction . The pare burden , 41, potoduction . Togethe+ DC activation in human malaria are sparse. In this study, we show that the absolute number of circulating CD16+ DCs are stable, and, moreover, their proportion in the total myeloid DC compartment increased during induced blood-stage malaria. This relative increase is likely a reflection of the loss of peripheral CD1c+ DCs [+ DC maturation during early subpatent Plasmodium infection is consistent with our recent report of CD16+ DCs being the only blood DC subset with increased HLA-DR expression during induced blood-stage P vivax malaria [Plasmodium species. High expression levels of HLA-DR are associated with efficient phagocytosis of P falciparum-infected RBCs [+ DCs likely enhances their potential to activate CD4+ T cells. Increased CD16+ DC maturation has been reported in P falciparum-sporozoite volunteer infection studies [Plasmodium infection by mosquito bite [P falciparum-sporozoite infection [+ DC maturation occurs in response to the early blood-stage of infection, independent of Plasmodium liver stages.Data relating to CD16D1c+ DCs . The inc malaria , suggestted RBCs  and esseted RBCs , togethe studies , which mito bite . Similarnfection , and dat+ DCs in adults with acute malaria when compared with convalescent controls. These findings are consistent with reports of reduced HLA-DR expression during acute malaria on CD1c+ DCs and CD141+ DCs in adults [+ DC maturation in children compared with adults seen here suggest that CD16+ DC dysfunction may result from cumulative exposure. Differences in CD16+ DC maturation between primary induced infection in naive adults and naturally acquired acute malaria are not surprising, and many factors may influence these differences including the magnitude of parasite burden or prior malaria. Dendritic cell maturation can be suppressed in the presence of IL-10 [P falciparum infection [+ DC suggest that increasing autocrine IL-10 production [In contrast to primary subpatent infection, HLA-DR and CD86 expression were reduced on CD16n adults  and chiln adults . In chilof IL-10 , 47. We of IL-10 , whereasnfection . These doduction  or increPlasmodium parasites on human blood CD16+ DC function. We demonstrate that CD16+ DCs are uniquely activated by P falciparum stimulation and significantly increase spontaneous coproduction of TNF and IL-10 during a primary subpatent P falciparum infection. Our data, presented here and previously [+ DCs are the only DC subset activated during induced blood-stage malaria. As dual cytokine producers, CD16+ DCs contribute to inflammatory as well as regulatory innate immune processes and are key responders during early subpatent P falciparum infection.Taken together, we describe for the first time the effect of eviously , 24, togThe Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at jiy555_suppl_Supplementary_FiguresClick here for additional data file.


The parasitic disease malaria threatens more than 3 billion people worldwide, resulting in more than 200 million clinical cases and almost 600,000 deaths annually. Vaccines remain crucial for prevention and ultimately eradication of infectious diseases and, for malaria, whole sporozoite based immunization has been shown to be the most effective in experimental settings. In addition to immunization with radiation-attenuated sporozoites, chemoprophylaxis and sporozoites (CPS) is a highly efficient strategy to induce sterile protection in humans. Genetically attenuated parasites (GAP) have demonstrated significant protection in rodent studies, and are now being advanced into clinical testing. This review describes the existing pre-clinical and clinical data on CPS and GAP, discusses recent developments and examines how to transform these immunization approaches into vaccine candidates for clinical development. Disease-limiting immunity that develops after multiple malaria episodes does not prevent parasitemia or transmission [Malaria is a life-threatening, multi-organ disease caused by blood stage infections with smission . WHO estsmission . Furthersmission .Anopheles vector, linked by short but pronounced population bottlenecks during host transitions. One such bottleneck occurs during transmission, when female Anopheles mosquitoes inoculate tens to hundreds of motile sporozoites into the human host. Following active penetration into the blood circulation, sporozoites are passively carried to the liver where they rapidly invade hepatocytes. Within a week, a single, clinically silent round of replication from a single P. falciparum sporozoite will give rise to 1 × 103 to 5 × 104 invasive forms of the parasite, termed merozoites, which initiate the pathogenic intra-erythrocytic replication cycle and can result in up to 1013 parasites at the height of an infection. The small number of naturally transmitted sporozoites in an injected inoculum may explain the apparent lack of acquired pre-erythrocytic immunity even in individuals with substantial anti-blood stage immunity (as evidenced by recurrent asymptomatic and low-density blood stage re-infections) [Malaria parasites are unicellular eukaryotes with a complex life cycle characterized by developmentally distinct phases of rapid and massive asexual replication in the vertebrate host and the before, during or shortly after liver stage development induce immunity targeting this clinically silent phase of the Plasmodium life cycle [Experimental malaria vaccine approaches using live sporozoites and different strategies to arrest the infection fe cycle . The confe cycle ,7,8. Whife cycle . In briefe cycle –13.There are four approaches to whole organism immunization: radiation-attenuated sporozoites (RAS), chemoprophylaxis and sporozoites (CPS), genetically attenuated parasites (GAP) and chemically attenuated parasites. In this review we will discuss novel insights and ideas principally on CPS and GAP, whereas RAS is discussed in more detail elsewhere in this special issue.2P. falciparum-infected mosquitoes while receiving CQ prophylaxis [Administration of sporozoites under chemoprophylaxis as an immunization method was first explored in rodent malaria models in the late 1970s–early 1980s using infection under chloroquine (CQ) prophylaxis –16. The phylaxis . This rephylaxis . Furtherphylaxis . These rphylaxis . Althougphylaxis ,21–24.−15 mosquito bites are required for >80% protection [Despite being logistically challenging to deliver, the dose of parasites required to generate immunity similar to RAS is considerably (orders of magnitudes) lower as just 3 × 10otection  comparedotection . These e2.1Using drugs targeting blood stages for CPS immunization has the advantage that full liver stage development can be achieved (as discussed in more detail later). There are many antimalarial drugs available that are candidates for CPS. They vary in efficacy, safety and mechanisms of action but only a handful have been evaluated in animal or clinical studies for use in CPS.Plasmodium liver stages such as primaquine, sulfadoxine–pyrimethamine, atovaquone–proguanil, doxycycline, azithromycin and clindamycin  could be used [Drugs that suppress blood stage growth can block infections during the first round of replication inside red blood cells. The exact mechanism of one such drug, CQ, remains under debate but most likely acts by disrupting detoxification of heme, a harmful by-product of hemoglobin digestion during the trophozoite phase of blood stage infection . Mefloqu be used . Primaqu be used .Thus the quiver of antimalarials available for potential use in CPS is well stocked. Although only CQ and MQ have been demonstrated effective in humans, alternative protocols have been demonstrated feasible in murine models using primaquine, pyrimethamine, artesunate, azithromycin, clindamycin and piperaquine ,29–32. M3Plasmodium [Plasmodium genome sequence of multiple species and through transcriptional profiling. The latter identified genes that are differentially up-regulated in sporozoites and liver stages, and therefore are most likely to be essential for parasite development through the liver [Parasites can be attenuated by deletion of genes that are necessary at different phases of liver stage developmental progression. This attenuation strategy was made possible by the development of gene deletion methodologies in asmodium ,35, delihe liver ,36,37 wiP. falciparum GAP showed considerable but incomplete attenuation [Genetically attenuated parasites (GAP) have demonstrated substantial to complete attenuation and sustained protective immunity in rodent malaria studies ,39. The enuation , indicatGAP are parasites with well-characterized genetic changes, while in RAS the effect of radiation acts upon the parasites’ DNA inducing non-specific damage that completely prevents parasite replication. Knowing the exact genotype allows precise characterization of the product, and provides manufacturing and regulatory advantages. Moreover, the timing of parasite arrest can be controlled in GAP, whereas RAS do not develop beyond the stage where DNA-replication is required. Consequently, GAPs that develop further into liver stage development may generate more potent immune responses. Indeed studies in rodent models with attenuated parasites have shown that immunization with greater numbers of parasites or with parasites that progress further into infection can provide superior immunity than low dose and/or early arresting parasites ,33,41. I3.1i.e. complete attenuation in the liver) and next it needs to be potent (i.e. generation of strong protective immunity). Therefore the foremost challenge is the selection of genes for deletion so that they create GAPs that are completely blocked in development in the liver.Two features are critical to the development of a candidate malaria vaccine consisting of attenuated parasites: first and most importantly it must be safe  from the genomes of rodent malaria parasites resulted in the creation of the first GAPs [Plasmodium 6-Cys protein family revealed that one member of this family, P52, was vital for parasite development in the liver and immunization of mice with sporozoites lacking P52 could also induce protective immunity [P. falciparum [p52−/p36−/P. falciparum GAP was conducted in 2008 where volunteers were exposed to 5 and then 200 bites from mosquitoes infected with this “double knockout” parasite. Attenuation appeared complete after exposure to 5 bites but following 200 bites, 1/6 volunteers became blood stage positive—indicating substantial but incomplete attenuation [rozoites ,37,43. Drst GAPs ,45. Moreimmunity . Mutantslciparum –49. The enuation . Recentlenuation ,51.P. falciparum GAP, only mutants lacking the gene sap1 , which encodes a protein involved in the regulation of transcripts in sporozoites, have been shown to fully attenuate liver stage development, even at very high sporozoite doses (up to 5 × 106 inoculated intravenously) [After the initial identification of the first rodent malaria parasite GAP, a number of additional gene deletion mutants have been identified that are completely or partially attenuated and induce protective immunity in mice ,52. Howeenously) –55. All enously) .slarp/sap1 has been removed in addition to a deletion of one or more of the 6-Cys genes from the P. falciparum genome. Specifically, the P. falciparum GAPs ‘p52−/p36−/sap1−’ and ‘Δb9Δslarp’ have both been examined in blood stage culture, in mosquitoes, in cultured human hepatocytes and in mice engrafted with human liver tissue and no evidence for full maturation in the liver was found [Consequently, GAP that are most advanced and currently ready for clinical evaluation consists of mutants where as found ,56. ThesP. falciparum GAPs are expected to arrest early in hepatic development, soon after invasion of hepatocytes by sporozoites. However, it has been demonstrated that immunization of mice with late liver stage-arresting GAP, lacking expression of a protein involved in fatty acid synthesis, FabB/F, elicits broader and more potent CD8T cell responses when compared to irradiated (early arresting) sporozoites [P. falciparum parasites lacking FabB/F expression fail to produce sporozoites, and thus are currently not useful for human immunization [The current rozoites . This elrozoites ,12. Thesnization .3.2P. falciparum genome is a complicated process that has required the targeted integration of plasmid constructs into the parasite genome by double crossover homologous recombination. In order to create P. falciparum GAPs for human use it is essential that any gene deletion is permanent and that reversion to the wild type genotype (and therefore phenotype) does not occur. Moreover GAPs must also be free of the drug-resistance markers that were used to select the deletion mutants, in order to meet regulatory concerns governing the use of genetically attenuated organisms as vaccines [p52−/p36−/sap1−’ and ‘Δb9Δslarp’ P. falciparum GAP strains fulfill all these requirements [The permanent removal of genes from the vaccines . The remvaccines ,59. Thesirements ,56 and a3.3i.e. an auxotroph), but importantly it is unable to develop in vivo in the absence of the compound. An example of such a strategy has been described where a mutant parasite has been generated that lacks a functional apicoplast and requires the addition of exogenous isopentenyl pyrophosphate for its survival in culture [The creation of a safe and protective GAP with multiple genes removed from the parasite genome could be considered a ‘first generation’ GAP. A ‘second generation GAP’ may include improvements to increase its immunogenicity, for example, one that arrests the parasite late into hepatic development and therefore induces stronger and broader protective immune responses as shown in rodent malaria models . Another culture .P. falciparum GAP potency could include (i) blood or transmission/mosquito stage Plasmodium antigens under the control of liver stage specific promoters to provide multi-stage immunity against malaria; (ii) genes that express proteins that could serve as adjuvants to enhance (CD8+) specific immune responses against liver stages; and (iii) genes encoding infection-limiting proteins (such as toxins) such that the GAP completely arrests late into liver stage development [Moreover, existing GAPs can be further engineered, as genetic modification also allows for the insertion of genes into the genome of attenuated parasites that have been modified to be more potent. Increasing GAP potency must be directed towards generating protective immune responses with the lowest number of parasites/dose and the fewest number of doses to generate sterile protection. Genes encoding molecules that could enhance elopment .4Plasmodium berghei and Plasmodium yoelii rodent models [http://www.anzctr.org.au/ACTRN12614000228684.aspx), but no results have been reported so far.In addition to radiation and genetically attenuated parasites for vaccination, sporozoites that have been incubated with centanamycin, a DNA-binding drug, also induce complete liver-stage arrest in both t models ,63. Immut models ,63. The t models . This st5Whole sporozoite vaccines face difficult hurdles in manufacturing, formulation and administration. However, they are far more effective than any alternative method investigated to date. Thus, there has been great interest in elucidating the immunological mechanisms underpinning sterilizing protection. This information can then potentially be used in the development and improvement of subunit vaccine strategies. Both cellular and humoral responses appear to play a role in protection induced by live sporozoite based immunization, although the relative contribution of each remains unclear.Early rodent studies using RAS demonstrated an essential and predominant role for CD8T cells with a contributing but dispensable role for antibodies ,65. Howein vitro [in vitro and in humanized liver-chimeric mice against P. falciparum challenged via mosquito bite [in vitro [Antibodies have historically been considered dispensable for protection elicited by whole sporozoite immunization, based on studies principally performed in animal models ,73–76. Hin vitro . Immunizin vitro ,80. Thesito bite . Howeverito bite . Althougin vitro . Indeed,in vitro . These ain vitro   demonstration of heterologous protection, (ii) development of practicable regimens with injectable sporozoites and (iii) safety with regard to 100% prevention of potentially life-threatening breakthrough infections. For CPS the design of a drug regimen that supports single dose, simultaneous administration of both parasite inoculum and drug is also paramount. For GAP vaccines, the identification of Plasmodium genes that are critical to mid-late liver stage development and hence, the creation of late arresting P. falciparum GAP that are significantly more potent than early arresting GAPs would be a major improvement [The availability of GMP-grade, cryopreserved rovement ,52,87–89To meet the challenges of mass vaccination in countries where malaria is endemic it is necessary that any malaria-vaccine, in particular a live-attenuated vaccine, is safe, affordable, convenient (minimizing the number of immunizations) and it needs to generate long-lasting strain-transcending protective immunity ,90. Whil


Plasmodium falciparum malaria. Associations between these variants and uncomplicated malaria are less clear.Multiple red blood cell (RBC) variants appear to offer protection against the most severe forms of Data from a longitudinal cohort study conducted in 3 sub-counties in Uganda was used to quantify associations between three red blood cell variants Hb , alpha thalassaemia 3.7 kb deletion, and glucose-6-phosphate dehydrogenase deficiency A—(G6PD 202A genotype) and malaria incidence, parasite prevalence, parasite density (a measure of anti-parasite immunity) and body temperature adjusted for parasite density (a measure of anti-disease immunity). All analyses were adjusted for age, average household entomological inoculation rate, and study site. Results for all variants were compared to those for wild type genotypes.In children, HbAS was associated, compared to wild type, with a lower incidence of malaria , lower parasite density upon infection , and lower body temperature for any given parasite density . In children, HbSS was associated with a lower incidence of malaria  and lower parasite density upon infection . α−/αα thalassaemia, was associated with higher parasite prevalence in both children and adults . G6PD deficiency was associated with lower body temperature for any given parasite density only among male hemizygote children .RBC variants were associated with non-severe malaria outcomes. Elucidation of the mechanisms by which they confer protection will improve understanding of genetic protection against malaria. Plasmodium falciparum malaria. Studies conducted in numerous populations have consistently shown that people with HbAS have a 70–90% reduced risk of severe malaria [Multiple red blood cell (RBC) variants are associated with protection against severe  malaria –4. Simil malaria –7. The a malaria . Some st malaria , and oth malaria . Mechani malaria –17.While protective associations between certain RBC variants and severe malaria have been shown consistently , 18, assP. falciparum infection, and regular entomological surveys to quantify transmission risk.Data from three cohorts conducted in areas of low, moderate, and high malaria transmission intensity in Uganda was used to quantify associations between three red blood cell variants and uncomplicated malaria outcomes. Notably, the cohort studies included passive surveillance for symptomatic malaria, active surveillance for Prospective cohort studies were conducted at three sites. Study sites were Walukuba, Jinja District, a peri urban area in South Central Uganda with a low malaria transmission intensity  or number of infective mosquito bites per person per year = 2.8); Kihihi, Kanungu District, a predominantly rural area in south western Uganda with moderate malaria transmission (aEIR = 32); and Nagongera, Tororo District, a predominantly rural area in south eastern Uganda with a very high transmission intensity (aEIR = 310)  until inStudy procedures and follow up have been described in detail –38. BrieAnopheles mosquitoes collected per night multiplied by the proportion of mosquitoes containing sporozoites at each site.Household level EIR was estimated using data from entomologic surveys carried out concurrently with the cohort studies, as previously described . BrieflyPresence of three RBC variants, the sickle locus (rs334), − 3.7α− thalassemia and G6PD-G202A (rs1050828), was tested as previously described . BrieflyAll data were collected using standardized forms and subsequently entered into Microsoft access before transfer to STATA  and R for analyses . The datOf 1344 participants enrolled in the cohort studies, 1322 (98.4%) were included in the analyses and 782 of these (59.2%) were followed through the end of June 2016  among children and 0.31 episodes PPY among adults. Among children, HbAS was associated with a 22% lower incidence of malaria  and HbSS with an 83% lower incidence of malaria , compared to children with wild type genotypes  . On average, children with HbAS had lower parasite densities by a factor of 0.66  compared to children with wild type genotype. There were no significant associations between alpha thalassaemia or G6PD deficiency and parasite densities . In models adjusted for parasite density, parasitaemic children with HbAS had body temperatures that were on average 0.13 °C lower  at the time of the visit, compared to children with wild type genotype. There were no significant associations between alpha thalassaemia and body temperature among parasitaemic children. Upon stratification, parasitaemic male children that were G6PD hemizygotes had temperatures that were 0.19 °C lower  compared those with the wild type G6PD genotype. There were no significant associations between G6PD deficiency and temperature among female parasitaemic children  [This study used data from 1322 participants enrolled in three longitudinal cohorts in Uganda to investigate associations between RBC variants and several outcomes of uncomplicated  malaria , 19 and  malaria , 19, 43.PfEMP-1) , 44. TheIn contrast to results for HbS, there were no protective associations between alpha thalassaemia and any of the outcomes measured. In fact, although α−/αα thalassaemia has been associated with protection against both severe , 18, 22 P. falciparum infection. Thus, G6PD deficiency may be associated with increased ability to tolerate high parasite densities without developing fever. However, the protective effect against symptomatic malaria was seen only in males, and not in heterozygous or homozygous females. There is considerable evidence to suggest that G6PD protects both male [Interestingly, the results from this study suggest that G6PD deficiency may play a role in anti-disease immunity. For any given parasite density, G6PD hemizygous parasitaemic male children had lower temperatures than wild type parasitaemic male children upon oth male  and femaoth male , 46, 47 oth male , 24.Results for associations between G6PD deficiency and uncomplicated malaria remain inconclusive.P. falciparum antibodies. Finally, there were very few cases of severe malaria, precluding exploration of associations between host polymorphisms and severe malaria.This study had several limitations. First, sample sizes were small for certain groups, notably the SS and G6PD homozygous genotypes, which limited the ability to evaluate their associations with malaria outcomes. However, these genotypes occurred at expected frequencies in these populations and sample sizes would be prohibitively large to study them in a cohort. Second, parasitaemia was measured using microscopy, which is much less sensitive than molecular techniques for identifying parasites –50. ImpaIn areas endemic for falciparum malaria, RBC variants may play an important role in protecting children against malaria. First, both HbAS and alpha thalassaemia are associated with decreased incidence of severe malaria. These strong associations probably explain the persistence of these balanced polymorphisms in human populations at risk of falciparum malaria. Second, some polymorphisms also appear to protect against uncomplicated malaria. Identified associations with HbAS provide the strongest evidence of protection, with evidence for lower risks of malarial incidence and enhancement of both anti-parasite and anti-disease immunity. G6PD deficiency may provide additional protection against progression to symptomatic disease, with evidence for enhanced anti-disease immunity. While the mechanisms by which common RBC variants protect against malaria remain incompletely understood, their associations with protection against malaria underline the exceptional selective pressure of malaria on the human genome.Additional file 1: Table S1. Prevalence of red blood cell variants stratified by study site and age group.


Is malaria infection during pregnancy associated with fetal head growth?In 2 cohort studies of 4291 pregnancies, falciparum malaria during pregnancy was significantly associated with the occurrence of decreased head circumference in newborns. Placental malaria characterized by increased placental syncytial nuclear aggregates, leukocyte infiltration, and imbalanced angiogenic factors was associated with the incidence of decreased head circumference.Plasmodium falciparum infection during pregnancy was associated with altered fetal head development, with possible consequences for fetal neurologic development. Malaria during pregnancy is associated with adverse events for the fetus and newborn, but the association of malaria during pregnancy with the head circumference of the newborn is unclear.To investigate the association of malaria during pregnancy with fetal head growth.Two cohort studies were conducted at the general maternity hospital of Cruzeiro do Sul  in the Amazonian region. One cohort study prospectively enrolled noninfected and malaria-infected pregnant women who were followed up until delivery, between January 2013 and April 2015. The other cohort study was assembled retrospectively using clinical and malaria data from all deliveries that occurred between January 2012 and December 2013. Data analyses were conducted from January to August 2017 and revised in November 2018. Clinical data from pregnant women and anthropometric measures of their newborns were evaluated. A total of 600 pregnant women were enrolled through volunteer sampling (prospective cohort study), and 4697 pregnant women were selected by population-based sampling (retrospective cohort study). After application of exclusion criteria, data from 251 (prospective cohort study) and 232 (retrospective cohort study) malaria-infected and 158 (prospective cohort study) and 3650 (retrospective cohort study) noninfected women were evaluated.Malaria during pregnancy.The primary end point was the incidence of altered head circumference in newborns delivered from malaria-infected mothers compared with that from noninfected mothers. Secondary end points included measures of placental pathology relative to newborn head circumference.Plasmodium vivax, Plasmodium falciparum, or both. Among 3882 newborns in the retrospective cohort study, 232 were born from mothers that had malaria during pregnancy. The prevalence of newborns with a small head (19 [30.7%] in the prospective cohort study and 30 [36.6%] in the retrospective cohort study) and the prevalence of microcephaly among newborns (5 [8.1%] in the prospective cohort study and 6 [7.3%] in the retrospective cohort study) were higher among newborns from women infected with P falciparum during pregnancy. Multivariate logistic regression analyses revealed that P falciparum infection during pregnancy represented a significant risk factor for the occurrence of small head circumference in newborns . Placental pathologic findings corroborated this association, with more syncytial nuclear aggregates and inflammatory infiltrates occurring in placentas of newborns born with decreased head circumference.In total, 4291 maternal-child pairs were analyzed. Among 409 newborns in the prospective cohort study, the mothers of 251 newborns had malaria during pregnancy, infected with This study indicates that falciparum malaria during pregnancy is associated with decreased head circumference in newborns, which is in turn associated with evidence of placental malaria. This cohort study conducted in the Amazonian region of Brazil investigates whether an association exists between malaria infection during pregnancy and fetal head growth assessed by head circumference of newborns. Therefore, maternal malaria is associated with major complications among fetuses and newborns, including being the primary cause of low birth weight, intrauterine growth retardation, abortion, stillbirth, premature delivery, and fetal death in malaria-endemic countries.1 In addition, in utero exposure to malaria parasites has been associated with a decreased head circumference (HC) among fetuses and newborns, a proportional decrease in size as an outcome of the intrauterine growth retardation.6Having malaria during pregnancy, especially falciparum malaria, can be devastating and fulminant, leading to high mortality rates for both mother and fetus, with approximately 125 million pregnancies at risk of infection each year.7 A group of microorganisms termed TORCH, which now also includes hepatitis virus, HIV, and most recently Zika virus, are frequently associated with decreased HC in newborns.9Although the brain insult is defined by the cranium size, it also reflects a reduction of brain volume and an impairment of cognitive abilities.10 Thus, to investigate the association of having malaria during pregnancy with fetal head growth, we analyzed data from both a prospective and a retrospective cohort of newborns who were delivered between 2012 and 2015 in the far western Brazilian Amazonian region.Several studies have reported the association of intrauterine infections with a high risk of low birth weight and brain injury among newborns.2, predominantly rainforest, and containing a population of approximately 200 000 inhabitants . This article was written according to the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cohort studies. All authors agreed to maintain the confidentiality of the data collected from the medical records and databases by signing the Term of Commitment for the Use of Data from Medical Records. Ethical approval was provided by the committees for research of the University of São Paulo and the Federal University of Acre , according to the Brazilian National Health Committee. All study participants or their legal guardians (if minors) provided written informed consent.Two cohort studies were conducted in the Amazonian region of the Alto do Juruá valley  between January 2012 and April 2015, evaluating data from maternal-child pairs for births occurring in the general maternity ward at Hospital da Mulher e da Criança do Juruá (Cruzeiro do Sul), where approximately 90% of total deliveries in the region occur. The Alto do Juruá valley is in the extreme southwest of the Brazilian Amazon Basin, covering an area of 74 965 kmP falciparum or Plasmodium vivax or were noninfected were followed up until delivery. During this period, all pregnant women diagnosed as having malaria by the local endemic surveillance teams were invited to participate in the study. Women who were initially considered noninfected were recruited during their first visit to the antenatal care clinic. Each woman was followed up by a trained nurse, which involved at least 2 domiciliary visits, at the second and third trimester, to monitor the woman’s clinical state in addition to the usual antenatal care.In total, 600 pregnant women who were enrolled between January 2013 and April 2015 through volunteer sampling and who were infected with At the time of recruitment, data were collected on socioeconomic, clinical, and obstetric variables, and peripheral blood and thick and thin blood smears were used to diagnose and confirm malaria infection. During the domiciliary visits, clinical and obstetric data were obtained and a peripheral blood sample was collected. An additional blood sample was collected for each episode of malaria during pregnancy. At the time of delivery, clinical data were collected from the mother, newborn, a placental fragment, and blood samples.15 An individual was considered within normal reference head circumference (NHC) range if their HC was within 1 SD of the mean. Newborns with an HC less than 1 SD below the mean were considered to have a small head (SH),16 and newborns with an HC less than 2 SDs below the mean were classified as having microcephaly.10 Detailed procedures for data collection, sample processing, malaria screening and treatment, other infectious agent screening, angiogenic factors and leptin levels, and newborns’ anthropometric measurements are given in the eAppendix in the The gestational age was estimated using the woman’s last menstrual period and adjusted using ultrasonographic data during the first trimester of pregnancy. Based on the gestational age, the HC, and sex, each newborn was assigned to groups using the definitions of the Intergrowth-21st Project.In total, 4697 maternal-child pairs were selected retrospectively through a population-based sampling of all deliveries occurring between January 2012 and December 2013. The data from the Brazilian Epidemiological Surveillance Information System (SIVEP)–Malaria on maternal malaria infection status during pregnancy were assembled with the clinical and anthropometric data presented in the medical records of the mother and the newborn. This was followed by the collection and collation of data to evaluate the newborns further . Newborns with an HC less than 1 SD below the median were considered to have an SH .16 Newborns with an HC less than 2 SDs below the median were classified as having microcephaly .17 Detailed procedures on malaria and other infectious agent screening, malaria treatment, and newborn anthropometric measurements are given in the eAppendix in the The gestational age was established by the woman’s last menstrual period, which was obtained from medical records. On the basis of the estimation of gestational age, HC, and sex, each newborn was assigned to groups using the World Health Organization child growth standards.Our analysis was restricted to newborns who had been born at term (37-42 weeks of gestation) with at least 2500 g of weight, as a single birth, and from mothers of fertile age (13-47 years old). Women were excluded if they had a history during pregnancy of smoking, drug use, or alcohol consumption or presented with other infections  or other comorbidities   with 95% CIs were estimated using a multivariate logistic regression approach. These models included infection by malaria (no or yes), maternal age (≥18 or ≤17 years old), and the number of gestations (≥2 or 1) as explanatory variables and SH (yes or no) or microcephaly (yes or no) as response variables. The first category for each explanatory variable was considered the reference.19 Missing data were imputed or filled in within a multiple imputation framework using the MICE library within R software.21 In particular, in the retrospective cohort study, 5 data sets were completed, and the results were pooled across, allowing for the uncertainty in the imputation process.Data were analyzed from January to August 2017 and revised in November 2018 using R (The R Foundation), Stata (StataCorp), and GraphPad Prism software. Continuous variables were summarized using mean (SD) values as well as median values and interquartile ranges (IQRs). Categorical variables were summarized using frequencies and percentages. Differences between groups were evaluated using the nonparametric Kruskal-Wallis test followed by the Dunn post hoc multiple comparison test and the Mann-Whitney test as appropriate. Categorical data and proportions were analyzed using χP vivax, P falciparum, or both (mixed)  met the inclusion criteria . Among t (mixed) . There w (mixed) .P = .005)  deviated from that in the noninfected group   A. To ens = .005)  or with P = .02) B, indicaP vivax infection, mixed infection, or P falciparum infection. The prevalence of newborns with an SH was higher among newborns from women infected with P falciparum during pregnancy (19 [30.7%]). Similarly, the prevalence of microcephaly doubled when a P falciparum malaria infection occurred (5 [8.1%])  or microcephaly  among the newborns . None of the newborns included in the association analysis tested positive for TORCH infections, syphilis, HIV, dengue, chikungunya, or Zika virus  presented with an SH, including 15 (3.7%) with microcephaly C. The ev [8.1%]) C. A multnewborns C. InfectP falciparum infection with the occurrence of an SH. Mothers whose newborns had an SH  experienced their first P falciparum infection later in gestation  than mothers whose newborns had an NHC . Moreover, much of the placental malaria manifestation in newborns with an SH  or microcephaly  could be associated with a past P falciparum infection compared with that for P falciparum–associated NHC (38 of 80 [48%])  .P falciparum–infected groups. Of note, compared with that for the noninfected group, we observed higher median numbers of infiltrated monocytes for all P falciparum–infected groups   and the P falciparum–microcephaly group   B. HoweveP = .02) D. MoreovP = .008)   remained to be evaluated, of which 232 were born from mothers who had malaria during pregnancy . OverallP falciparum–infected mothers   had an SH and 161 (4.2%) had microcephaly. In the retrospective cohort study, similar to the prospective cohort study, the prevalence of newborns with an SH was more than one-half times as high (36.6%), and microcephaly was doubled (7.3%), among newborns with P falciparum during pregnancy is significantly associated with the occurrence of decreased HC in newborns and, to some extent, with microcephaly. The finding on the HC decrease is independent of the currently known association between malaria with overall fetal growth because low-birth-weight and preterm newborns were deliberately excluded from our analysis. An increased risk for decreased HC associated with P falciparum infection was supported by both our prospective cohort study  and our retrospective cohort study . These observations reinforce the knowledge that having malaria during pregnancy increases the risk of problems in fetal development.22Malaria during pregnancy increases the risk of adverse fetal outcomes. This study shows evidence that infection with P falciparum infection may be a mechanism contributing to the decreased fetal head growth. This hypothesis was supported by the observed histopathologic alterations combined with an imbalance in angiogenic factor production in placentas from newborns with congenital SH or microcephaly born to P falciparum–infected mothers. Local inflammation can generate a setting of hypoxia or ischemia that would alter the transportation of both nutrients and respiratory gases to the unborn fetus, which can result in cranial malformation owing to an inadequate supply of nutrients and oxygen.23 Oxidative stress caused by hypoxia also leads to structural and functional alterations in intrauterine development.24 This scenario is often observed in cases of placental malfunction due to various etiologies and to prolonged or premature labor.25We hypothesize that the placental inflammatory process acting against the 26 were markedly increased in placentas from newborns with decreased HC. In addition, SNA has been repeatedly observed in placentas from P falciparum–exposed women.28 The placental alterations observed in this study, including the increased SNA and monocyte infiltration, are consistent with previous reports on the response to placental P falciparum–sequestering parasites, which characterizes placental malaria development.28 It was unsurprising that infection with P vivax was not associated with a decreased HC phenotype because this parasite is known to not sequester in the placenta. Previous studies have demonstrated that P vivax infection induces less of a placental inflammatory process compared with that induced by P falciparum infection.28The values of SNA, which have been associated with intrauterine growth retardation caused by local hypoxia or oxidative stress,P falciparum–SH group, few observed differences reached statistical significance in this study, possibly owing to the small sample size of this group, but the overall placental malaria phenotype was more prominent and widespread than that in the noninfected and P falciparum–NHC groups. Nevertheless, our observations reflect only a picture at the moment of birth, and it is unclear how placental alterations may be associated with the development of the fetus.Regarding the P vivax. However, P falciparum is transmitted in specific regions, including the Alto do Juruá valley, where it is responsible for 46% of the total malaria infections in Brazil.13 Despite Brazil being an area with low transmission for malaria, effective control strategies, and early treatment provision, we observed adverse events in newborns similar to those reported in areas of high endemicity.Currently, much of what is known about falciparum gestational malaria is based on studies performed in high-transmission areas in Africa, which, in general, are settings that have precarious health systems and inadequate or late treatment provision. In Brazil, approximately 85% of malaria infections are caused by 10 Recently, other studies that have evaluated newborns retrospectively in different Brazilian regions have also reported a higher prevalence of microcephaly in newborns before the Zika outbreak.30 It is puzzling that the United States, with nearly the same number of births per year as Brazil, reports approximately 25 000 newborns with microcephaly yearly, whereas Brazil reported only 150 before the Zika epidemy.32 These observations indicate an inconsistency of the data released by the Brazilian authorities, likely owing to underreporting.Surprisingly, the prevalence of microcephaly observed by us is far higher than what has been previously reported by the Brazilian Ministry of Health.P falciparum infection, we had access to only a few placentas. This small sample size limited our statistical analysis; however, most of the factors analyzed indicated intensified placental malaria compared with placentas from newborns with an HC within the normal reference range.Our work has some potential limitations. First, the HC among newborns was assessed only at birth because morphometric measurements through ultrasonography during pregnancy, as well as the possibility of acquiring newborn head imaging, were not possible. Second, decreased HC has different etiologies, namely, genetic causes and actions of infectious agents. Although we removed some confounding factors, such as TORCH infections, syphilis, HIV, dengue, chikungunya, Zika virus, and other comorbidities, studies to detect genetic abnormalities among the patients were not performed. Third, although in both the prospective cohort study and the retrospective cohort study the logistic regression analyses indicated a clear association between an SH and P falciparum infection during pregnancy was associated with decreased HC and, in extreme cases, with microcephaly. The consequences of gestational malaria throughout fetal neurologic development, which can lead to poor neurocognitive and behavioral development, represent serious long-term health problems. Physicians should periodically assess the development and academic achievements of these children, with a comprehensive neurocognitive evaluation, to guide preventive and rehabilitative assistance that might improve outcomes. Extensive epidemiologic prospective studies, involving the collection of biological, clinical, and socioeconomic data and potential confounding factors, are required to establish the prevalence of newborns with an SH and the prevalence of newborns with microcephaly and their association with malaria. The evidence of the association observed in our study supports an urgent need to protect pregnant women and their unborn children from malaria infection.This work provided evidence that


Plasmodium vivax infection is rising in sub-Saharan Africa, where Plasmodium falciparum is responsible for more than 90% of malaria cases. While P. vivax is identified as a major cause of severe and cerebral malaria in South east Asia, the Pacific and South America, most of the severe and cerebral cases in Africa were attributed to P. falciparum. Cases of severe malaria due to P. vivax are emerging in Africa. A few severe P. vivax cases were reported in Eastern Sudan and they were underestimated due to the lack of accurate diagnosis, low parasitaemia and seldom use of rapid diagnostic tests (RDTs).P. vivax cerebral malaria based on the positive blood film and the amplification of P. vivax specific 499 bp amplicon using Plasmodium multi-species multiplex Polymerase Chain Reaction (PCR). The patient was treated with quinine 10 mg/kg body weight for 10 days followed by primaquine 15 mg/days PO for 2 weeks. The symptoms subsided within 48 h and the patients was cured and released from the hospital.A 60-year-old Sudanese male presented to the Al Kuwaiti hospital in the Sudan capital Khartoum. On admission, the patient was complaining of fever (measured temperature was 38 °C), sweating, chills, vomiting and confusion in the past 2 days prior to his admission. He rapidly deteriorated into a coma state within 48 h of the admission, with significant neck stiffness. He was admitted to the intensive care unit and was suspected of meningitis. Lumbar puncture was not performed since the patient was suffering from spinal cord disc. Brain CT scan was unremarkable. Several biochemical, haematological tests, and blood film for malaria were performed. The results of the laboratory tests were within the normal range except of mild elevation of the total white blood cell count and a significant decrease in the platelets count. Malaria parasites were seen in the blood film with high parasitaemia (quantified as 3 +++). The patient was diagnosed as Plasmodium vivax is an emerging cause of cerebral malaria in adults in Sudan and should be considered in the differential diagnosis of cerebral malaria for proper management of patients. Plasmodium vivax infection is geographically widely spread. In 2013, it comprised 8% of all estimated malaria cases globally, and represented 47% of cases outside sub-Saharan Africa, and < 1% of cases in the WHO African Region [P. vivax cases in 2013, with the total number of malaria deaths that could be attributed to P. vivax ranging between 1900 and 10,000 globally.P. vivax malaria were reported from all endemic regions. In 2015, severe vivax malaria was attributed to cause 16% of all malaria related mortality outside sub-Saharan Africa [P. vivax malaria was estimated ranging between 0.012 and 0.063%, while the risk of severe disease was estimated between 0.29 and 0.82% [Severe cases and deaths due to Plasmodium vivax infection is an emerging public health problem in Sudan with an overall prevalence of 26.6% among malaria cases in different regions of the country [P. vivax cases were mostly neglected and misdiagnosed as Plasmodium falciparum infection or confused with other fever causing disease. There were a few reports of severe P. vivax malaria cases in Sudan, suggesting an underestimation of the prevalence of severe P. vivax infection due to the low sensitivity of the currently used blood films and the seldom use of rapid diagnostic tests (RDT) [ country . Severe ts (RDT) –6.P. vivax infection for differential diagnosis form P. falciparum cerebral cases for effective management of patients.There is a need for accurate and rapid diagnosis of severe A 60-year-old Sudanese male presented to the Al Kuwaiti hospital in the Sudan capital Khartoum complaining of fever (measured temperature was 38 °C) associated with sweating, chills, vomiting and confusion for 2 days prior to admission. He had been diagnosed with poorly managed diabetes mellitus for 20 years, and he was on Insulin Mixtard 30/70.3.On his admission, the patient did not complain of any gastrointestinal Tract (GIT), urinary or respiratory disorders, but was very ill and feverish. Within 48 h post admission he rapidly deteriorated into a coma state; Glasgow coma scale of 6 and did not react to stimuli with significant neck stiffness. He was admitted to the intensive care unit and was suspected of meningitis. Lumbar puncture was not performed since the patient had spinal cord disc prolapse. Brain CT scan was unremarkable. Several biochemical and haematological tests and blood film for malaria were performed. The renal and liver parameters were within the normal range. The patient had a fasting blood sugar of 295 mg/dl, haemoglobin of 11 g/dl, a platelet count of 43,000 cells/mm3, erythrocyte sedimentation rate (ESR) 18 mm, and a total white blood count of 13.4000 cells/mmP. vivax (quantified as 3 +++). PCR amplified P. vivax specific band of 499 bp using Plasmodium multi species multiplex PCR primers (Fig. P. vivax cerebral malaria case. He was started on intravenous fluids, intravenous quinine at the dose of 10 mg/kg body weight IV for 10 days and Ceftriaxone 1 g IV PD for 7 days, followed by primaquine 15 mg/days PO for 2 weeks. The vital signs improved over the next 48 h. The patient was released from the hospital 12 days post admission.Blood film for malaria parasites was positive by light microscopy for ers Fig. . The patP. vivax infection is rising in Sudan and has increased from about 5% in 2013 to 26% of the total malaria cases [P. vivax cases were reported from many endemic areas in South East Asia, the Pacific islands and South America, a few cases were reported in Africa reflecting the neglected status of P. vivax in Africa [The incidence of P. vivax until recently. The fact that he was a known type 2 diabetic patients, presenting with high grade fever, chills, vomiting, sweating and confusion lead to suspicion of serious infections, including meningitis and falciparum malaria, which is the common cause of severe malaria in Sudan [P. vivax infection which was confirmed by the positive result of Plasmodium multi species PCR [P. vivax malaria reported in other endemic areas including those in children in eastern Sudan were accompanied by low parasitaemia contrary to the findings of this study [P. vivax malaria 3 months before his current illness. He presented with fever, chills and confusion. He was successfully treated with quinine 10 mg/kg body weight IV for 10 days, ceftazidime 1G IV 8 hourly for 7 days followed by primaquine 15 mg/days PO for 2 weeks evident by the negative blood film at the end of the treatment course. The recent attack could be a recurrent attack since P. vivax prevails at low parasitaemia making its detection difficult [P. vivax drug resistance in Sudan, however the possibility of noncompliance to primaquine treatment could be excluded strengthening the possibility of the persistent of the parasite from the first infection [The patient described in the report was living in Khartoum the capital city of Sudan where malaria transmission is seasonal and non-endemic of in Sudan . The rapin Sudan . Lumbar cies PCR . Interesis study . The facifficult . It is wP. vivax as a cause of cerebral malaria in adults in Sudan and recommends considering P. vivax in the differential diagnosis of cerebral malaria caused by P. falciparum that requires a different treatment regimen. Plasmodium vivax treatment should be closely supervised to assure cure of the infection.This report identified


Plasmodium falciparum malaria. However, there are limited studies on the effect of P. falciparum malaria infection on oxidative stress in SCD patients.Oxidative stress plays a vital role in the pathogenesis of both Sickle Cell Disease (SCD) and P.falciparum malaria. The biomarkers namely: malondialdehyde (MDA), reduced glutathione (GSH), catalase (CAT) and glutathione peroxidase (GPx) were determined in plasma samples from SCD malaria positive, malaria positive, SCD malaria negative and healthy control participants. The genetic diversity of P.falciparum was determined by nested polymerase chain reaction of merozoite surface protein-2 (MSP-2) gene.A cross-sectional study was undertaken to compare levels of biomarkers of oxidative stress in isolates from SCD patients with uncomplicated P < 0.0001). In contrast, the mean concentration of GSH (P < 0.0001) and GPx (P < 0.0001) were significantly lower in SCD malaria than SCD controls. Although not significantly different, the mean concentration of MDA was higher (P = 0.0478), but the geometric mean parasite density (P = 0.2430) and multiplicity of infection (P = 0.3478) were lower in SCD malaria samples than in malaria samples. The most prevalent MSP2 allelic family was IC3D7 in SCD malaria (72%) and Malaria (76%) samples. The biomarkers of oxidative stress were not significantly different between IC3D7 and FC27 allelic families of MSP2.Out of 207 participants, 54 (26%) were SCD malaria positive, 51 (24%) malaria positive, 51 (24%) SCD controls and 51 (24%) healthy control individuals. The mean concentration of MDA was significantly higher in SCD malaria positive than SCD controls (P.falciparum malaria.We identified severe oxidative stress in Sickle cell disease patients with uncomplicated  Plasmodium falciparum infections in sub-Saharan Africa . The lower MOI may be due to severe oxidative stress in SCD malaria. In constrast, a much lower overall MOI of 1.43 was found SCD patients with malaria in Nigeria [P.falciparum causes oxidative stress regardless of parasite strain.Of note, no studies have investigated genetic diversity of  Nigeria . The lowThis study had several limitations. First the study had a small sample size for each of the four groups (~ 50 each) which may have limted our power to identify certain effects. Secondly, TBARS method assay is known to have limitations in that it measures many different aldehydes and decomposition products from H2O2 which may translate into problems with specificity and variability of data. Thirdly, reticulocyte counts were not determined which could have provided useful information for preliminary assessment of hemolytic or dyserythropoietic anemia. More studies are needed to investigate these aspects. Nevertheless, our study provides novel insights into the effects of malaria on oxidative stress in SCD patients (HbSS).P.falciparum parasites [P.falciparum malaria.This study showed increased lipid peroxidation and reduced antioxidant levels in the SCD+/ malaria isolates than the SCD controls. Although not significantly different, the mean GMPD was lower in SCD+/ malaria patients than SCD−/malaria patients. Thus, erythrocytes from SCD patients are subjected to a double oxidative stress, the first stress due to the mutation and secondly stress induced by the developing arasites . These r


Plasmodium species. Solubilized hemozoin catalyzes the atom transfer radical polymerization of N-isopropylacrylamide above the lower critical solution temperature of poly(N-isopropylacrylamide). The solution becomes turbid, which can be observed by naked eye and quantified by UV-visible spectroscopy. The rate of turbidity increase is proportional to the concentration of hemozoin, with a detection limit of 0.85 ng mL−1. Malaria parasites in human blood can be detected down to 10 infected red blood cells μL−1. The assay could potentially be applied as a point-of-care test. The signal-amplification of an analyte by biocatalytic precipitation polymerization represents a powerful approach in biosensing.Methods to diagnose malaria are of paramount interest to eradicate the disease. Current methods have severe limitations, as they are either costly or not sensitive enough to detect low levels of parasitemia. Here we report an ultrasensitive, yet low-resource chemical assay for the detection and quantification of hemozoin, a biomarker of all  Plasmodium species) to catalyse the polymerization of N-isopropylacrylamide.Methods to diagnose malaria are of interest but can be costly or not sensitive enough to detect low levels of parasitemia. Here the authors report an ultrasensitive method by using hemozoin (a biomarker of all  Plasmodium species that is transmitted via mosquito bites. In 2017, the WHO  estimated 3.1 billion people at risk of being infected by malaria, with 1.1 billion people at high risk. 219 million cases, an increase of 3 million cases over the previous year, were reported ending in 435,000 deaths1. Africa remained as the region with the highest mortality burden accounting for 93% of the deaths. Sixty-one percent of the worldwide deaths were children under the age of five. Detection of malaria infections is one of the keys to eradication of the disease. For example, highly sensitive techniques to screen populations in endemic areas are needed to detect and subsequently treat asymptomatic individuals with submicroscopic Plasmodium sp. infections who do not display symptoms, but who represent a major reservoir for the transmission of the disease3. An effective field test for malaria must be highly sensitive, its reagents have to be robust to withstand transport and storage in tropical regions, it has to be easy to use and should be of low cost. Today, no single malaria field test on the market fulfills all these criteria. Microscopy in combination with Giemsa staining is the most widely used technique to diagnose malaria4. It allows the identification of the malaria species and has a detection threshold of ~50 infected red blood cells (iRBC) µL−15. However, this method requires well-trained microscopists to correctly identify the parasite in a blood smear. As a result, this diagnosis is mostly performed at central laboratories or hospitals. This is the reason why malaria rapid diagnostic tests (MRDTs) were developed for field use. Since the WHO recommended in 2010 the diagnosis of all suspected cases, the number of people using MRDTs has increased from <200,000 in 2005 to more than 314 million in 20147. There are more than 200 MRDTs currently available;8 most of them are based on immuno-chromatography strips which perform antigen–antibody complexation8. However, their sensitivity to malarial parasites is relatively low (detection limits around 200 iRBC µL−1)9. While these tests are sufficient to diagnose patients with malaria symptoms, they are not sensitive enough to identify asymptomatic carriers of malaria parasites. To identify these will be a stumbling block for regions where the disease is targeted for eradication. Moreover, current MRDTs are unable to quantify parasitemia, which would be beneficial to monitor treatment efficacy and transmission dynamics. Further deficiencies of MRDTs include cross reactions with antigens of other parasites or intrinsic lack of target antigen in the parasite population such as the reported deletion of the hrp2 gene, resulting in false positive or negative results10. Moreover, the antibodies used in the strips do not always perform as expected at the high and humid temperatures encountered in the regions where malaria is present8. As typical MRDTs should be kept below 30 °C11, an effective cooling chain must be implemented during their transport and storage. Alternatively, highly sensitive methods such as loop-mediated isothermal amplification12 or enzyme linked immunosorbent assay (ELISA)13 are also available, and efforts are underway to develop quantitative real-time polymerase chain reaction (qPCRs) for field work16. However, up to now, none of these techniques can be used as point of care tests. Few malaria diagnostic tests are based on the detection of hemozoin, which is a by-product of hemoglobin digestion by the malaria parasite18. It is a biocrystal which consists of centrosymmetric μ-propionate dimers of heme . The formation of poly(N-isopropylacrylamide) (PNIPAAm) can be simply detected by conducting the polymerization at 37 °C, i.e., under conditions in which the polymer precipitates49. This results in a transparent, colorless liquid becoming turbid . As some of the experiments required larger amounts of hemozoin, synthetic hemozoin  was also used. sHz is considered a chemical and structural analogue to nHz , which is insoluble under physiological conditions, was obtained from nHz Fig. 19.48. As the reactions proceeded, the polymers precipitated and the transparent solution turned turbid. This process was observable by bare eye  was defined as the quantitative read-out of the assay.Solubilized Hz can catalyze the polymerization of NIPAAm to PNIPAAm in the presence of the ATRP reagents sodium ascorbate (reducing agent) and 2-hydroxyethyl bromoisobutyrate (HEBIB) (initiator) Fig. . Polymereye Fig.  and was ime Fig. . At longA series of experiments was carried out to optimize the parameters for the assay. Reactions that were carried out in air did not result in a homogeneous turbidity throughout the solution or did not polymerize at all  (R2 = 0.98). At lower concentrations of the analyte, the signal scaled with the concentration of nHz, but not linearly. The sensitivity of the assay could be further increased by conducting the extinction measurements at 380 nm. The assay read-out at this wavelength increased linearly with the concentration of nHz at very low amounts of nHz  (1 iRBC µL−1 corresponds to ~0.6 pg µL−1 Hz), showing the potential for the development of a highly sensitive technique for malaria diagnosis based on Hz-catalyzed precipitation polymerization.If the hemozoin-catalyzed precipitation polymerization were to be used as a quantitative assay to determine hemozoin in a solution, the read-out of the assay should depend on the concentration of hemozoin. Therefore, precipitation polymerizations were carried out with various amounts of solubilized Hz Fig. . A higheoin Fig. . For natnHz Fig. . The dec52. Most likely, they reduce the catalytic polymerization activity of the heme because they are known to scavenge free radicals53.The dose-response curve for sHz is similar to the one of nHz . Upon Hz-catalyzed precipitation polymerization of NIPAAm above the LCST of the polymer, the reaction mixture becomes turbid. This can be seen qualitatively by unaided eye or recorded quantitatively by turbidity measurements. The rate of turbidity formation scales linearly with the concentration of solubilized hemozoin, which makes the assay quantitative. The assay can detect as low as 0.85 ng mLUnless otherwise stated, in a typical experiment 1.429 g (12.63 mmol) of NIPAAm were weighted together with 250 mg (1.26 mmol) of (+)-sodium L-ascorbate into a round-bottom flask. Thereafter, 10 mL of sodium phosphate buffer solution  were added to obtain a stock solution which was employed for the polymerization reactions. 0.700 mL of this stock solution were pipetted into disposable semi-micro poly(methyl methacrylate) cuvettes (path length: 1 cm). Various volumes of solubilized hemozoin solutions in 0.4 M NaOH were added and adjusted to a total volume of 50 μL of 0.4 M NaOH. The volume of buffer was adjusted to achieve a total volume of 950 μL in each cuvette with a pH of 6.9. The solutions were then sealed against ambient oxygen by adding 500 μL of mineral oil. The cuvettes were incubated for 3 min at 37 °C. Finally, the polymerization was initiated by the addition of 50 µL of a 375 mM 2-hydroxyethyl HEBIB solution in DMF. The final concentration of reagents in the assay was: 767 mM NIPAAm, 18.7 mM HEBIB and 76.7 mM sodium ascorbate, i.e., a ratio of 41:1:4.1. The cuvette was placed in a thermostatted cuvette holder (37 °C) of a UV–vis spectrometer and extinction was recorded at 600 nm or at 380 nm every 30 s with an integration time of 1 s for up to 4 h.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1


Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α “tag”). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or “parent” domain) coupled with the succeeding cysteine-rich interdomain region (CIDR).A protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season.Malian serologic responses to a PfEMP1’s DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure.Larger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure.The online version of this article (10.1186/s12936-019-2905-9) contains supplementary material, which is available to authorized users. Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens are critical to parasite sequestration, and their genetic diversity likely facilitates host immune system evasion. The var gene family encodes PfEMP1 antigens, whose extracellular binding region is made up of Duffy-binding-like (DBL) domains interspersed with cysteine-rich interdomain regions (CIDRs). A 300–400 variable nucleotide region surrounded by semi-conserved motifs in the DBL-α domain, the first N-terminal var domain, acts as a unique “fingerprint” specific to individual vars. DBL-α “tag” sequencing facilitates analyses of both PfEMP1 genetic diversity [iversity  and seroAntibody responses to locally derived DBL-α tags in Papua New Guinea increased in magnitude and prevalence with age in children, suggesting that DBL-α tags correlate with malaria exposure and potentially the development of natural immunity to malaria . In contP. falciparum exposure. In addition, seroreactivity to the DBL-α tag was not predicted to correlate with seroreactivity to the parent DBL-CIDR head structure, given the head structure’s additional structural components and its relative sequence conservation.To identify accurate markers of malaria exposure, serologic responses to the DBL-α tag of a PfEMP1 and its constitutive domains were measured. Serum samples from malaria-exposed Malian children, aged 1 to 6 years, and adults were studied on a custom PfEMP1 microarray populated with DBL-α tags, parent DBL-CIDR head structures (i.e. the entire DBL-α domains encompassing the tags with the succeeding CIDR domains), and downstream PfEMP1 fragments. The hypothesis was that seroreactivity to DBL-α tags among malaria-exposed individuals increases with age and with cumulative P. falciparum open reading frame, (2) in vivo recombination cloning, (3) in vitro transcription/translation (IVTT), and (4) microarray chip printing [Protein microarray construction followed a four-step process that included: (1) PCR amplification of each complete or partial printing . This IVprinting . The micThe protein microarrays were probed with sera from 18 adults aged 18–55 years old in the control arm of a trial of an apical membrane antigen 1 (AMA1) vaccine adjuvanted to GlaxoSmithKline’s AS02A (FMP2.1/AS02A)  using puAll participants or guardians of participants provided written informed consent, and the trial was conducted under the Declaration of Helsinki. The institutional review boards of the Faculty of Medicine, Pharmacy and Dentistry, Bamako, Mali, and the University of Maryland approved the study protocol.Seroprevalence is the proportion of serum samples that recognized a protein fragment. A serum sample recognized a protein fragment if the fluorescence intensity was greater than two standard deviations above the malaria-naïve control mean for that fragment . Group “rs. P-values were two-sided, and α = 0.05. No adjustment was made for multiple comparisons as per previous protein microarray analyses [Change in seroreactivity for a protein fragment between the pre- and peak season for children and pre- and post-transmission season for adults was determined with a Wilcoxon signed-rank test for matched samples. Correlation between seroreactivity of DBL-α tag and DBL-CIDR head structures was measured with the Spearman correlation coefficient, analyses , 12.Fluorescence intensities to DBL-α tags and their parent DBL-CIDR head structures were measured using sera collected from Malian children and adults and North American malaria-naïve adult controls Fig. . OverallComparing the seroprevalence of DBL-α tags and DBL-CIDR head structures, serorecognition of DBL-α tags did not consistently predict parent DBL-CIDR head structure serorecognition Fig. . For twoFor three PfEMP1 antigens, paediatric and adult sera demonstrated equivalent DBL-α tag serorecognition, but the proportion of DBL-CIDR head structure serorecognition was greater in adults versus children , but only one of the corresponding parent DBL-CIDR head structures also had increased seroreactivity  and to the corresponding DBL-CIDR domain was determined. Reactivity to a DBL-α tag did not correlate well with reactivity to the corresponding parent DBL-CIDR head structure Table . DBL-α tP. falciparum malaria. Parent DBL-CIDR head structures were better indicators of malaria exposure. Serologic prediction of malaria exposure may be better estimated by using larger PfEMP1 fragments, such as head structures that include conserved regions, as targets for serological testing.Serologic responses to a PfEMP1’s DBL-α tag region did not correlate with seasonal malaria exposure or with responses to the parent DBL-CIDR head structure in either adults or children from an area of Mali with intense seasonal exposure to DBL-α tags have been used as a unique “fingerprint” reflecting PfEMP1 diversity in gene expression, serologic, and population genomics studies , 13–15. These findings contrast with results from a study done in Papua New Guinea that found that serologic responses to DBL-α tags revealed differences in malaria exposure. One hundred twenty-three DBL-α tag sequences from Papua New Guinea populated a protein microarray that was used to seroprofile Papua New Guinea adults and children, revealing that children’s antibody responses peaked between four and 15 years of age . In thisAntibody responses to particular PfEMP1 domains have been associated with protection from clinical malaria in several studies , 18–20, P. falciparum genomes, DBL-α and DBL-β domains have more sequence conservation than other DBL domains [Earlier microarray studies have found that more conserved PfEMP1 fragments are markers of malaria exposure. In a study of Malian children and adults followed during the malaria season, the most differentially seroreactive fragments in adults compared to children contained the intracellular ATS domain, which is more conserved than extracellular domains , 21. Sim domains . Here, c domains , 24.Escherichia coli system and lack post-translational modifications such as glycosylation [Escherichia coli expression system has not been confirmed.This study had some limitations. The examined DBL-α domains were from the reference strain 3D7, not from circulating Malian parasites in the population. In spite of this, individuals had seroreactivity to head structures and to the downstream PfEMP1 fragments, particularly Malian adults, who as a group recognized all DBL-CIDR head structures and downstream fragments. This suggests that the PfEMP1 antigens from 3D7 may represent the much larger circulating PfEMP1 population in Mali to some degree. Another limitation is the limited DBL-α tag population studied: DBL-α tags from nine 3D7 PfEMP1 antigens were examined, which represents only a small subset of the 60 3D7 PfEMP1 antigens, although at least one member of each PfEMP1 subgroup was included. This limited representation of PfEMP1 antigens could be another possible explanation for the limited serorecognition by children. It is possible that DBL-α tags from other 3D7 PfEMP1 antigens could have elicited more serorecognition in Malian children, but were not represented on the microarray. Whereas proteins expressed on this microarray platform have been shown to induce antibody responses similar to those from purified proteins, these proteins are produced with an Plasmodium falciparum. In particular, they have recently been used to pinpoint epitopes targeted by antibodies in a DBL-α domain that mediates rosetting [To further examine the role of conserved PfEMP1 domains in inducing serologic responses, peptide microarrays are a potentially useful tool to identify reactive epitopes. Such an approach may provide insight distinct from protein microarrays, where entire domains are included and may elicit differential seroreactivity. A protein microarray does not permit identification of the specific roles of either more conserved or more diverse regions within a protein fragment. Peptide arrays have already been used as a tool for studying epitopes in variant surface antigen proteins in osetting  and epitosetting . This apMalian serologic responses to a PfEMP1’s DBL-α tag, a semi-conserved short fragment of the DBL-α domain, did not correlate with seasonal malaria exposure or responses to the parent DBL-CIDR head structure in either adults or children. Larger, more conserved PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences are thus well suited for study as serologic predictors of malaria exposure on protein microarrays. Further support for this finding would include testing a broader panel of PfEMP1 antigens, including those from non-3D7 reference strains and from endemic parasite genomes.Additional file 1. Sequences, classifications, and domains of the PfEMP1 antigens used to populate the custom protein microarray.