P. vivax and P. falciparum malaria in patients from the Brazilian Amazon.A proliferation‐inducing ligand (APRIL) and B cell activation factor (BAFF) are known to play a significant role in the pathogenesis of several diseases, including BAFF in malaria. The aim of this study was to investigate whether APRIL and BAFF plasma concentrations could be part of inflammatory responses associated with P. vivax and P. falciparum malaria patients (n = 52) resident in Porto Velho before and 15 days after the beginning of treatment and from uninfected individuals (n = 12). We investigated APRIL and BAFF circulating levels and their association with parasitaemia, WBC counts, and cytokine/chemokine plasma levels. The expression levels of transmembrane activator and calcium‐modulating cyclophilin ligand interactor (TACI) on PBMC from a subset of 5 P. vivax‐infected patients were analyzed by flow cytometry.Blood samples were obtained from P. vivax and P. falciparum infections whereas BAFF levels were only increased during acute P. falciparum malaria. Although P. vivax and P. falciparum malaria patients have similar cytokine profiles during infection, in P. vivax acute phase malaria, APRIL but not BAFF levels correlated positively with IL‐1, IL‐2, IL‐4, IL‐6, and IL‐13 levels. We did not find any association between P. vivax parasitaemia and APRIL levels, while an inverse correlation was found between P. falciparum parasitaemia and APRIL levels. The percentage of TACI positive CD4+ and CD8+ T cells were increased in the acute phase P. vivax malaria.APRIL plasma levels were transiently increased during acute These findings suggest that the APRIL and BAFF inductions reflect different host strategies for controlling infection with each malaria species. Plasmodium species that infect humans, P. falciparum and P. vivax are responsible for 95% of malaria cases around the world. Although P. falciparum accounts for the vast majority of morbidity and mortality, P. vivax has a wider geographic distribution and causes significant symptomatic disease Plasmodium spp. Malaria is one of the most important human parasitic diseases. Nearly half the world's population is at risk of contracting malaria, with an estimate global annual incidence of about 212 million clinical cases and almost 429,000 deaths The mechanisms regulating BAFF and APRIL‐system molecule expression are poorly known. However, it is recognized that cytokines such as interferon (IFN)‐γ, IFN‐α, TNF, and Interleukin (IL)‐10 as well as granulocyte colony‐stimulating factor (G‐CSF), CD40 ligand (CD40‐L), lipopolysaccharides, and peptidoglycans can upregulate BAFF or APRIL expression in different cells P. falciparum malaria, BAFF levels are increased in plasma samples from infected children in Kenya P. falciparum antigens and hemozoin enhanced BAFF surface expression as well as secretion by human monocytes and increased B cell proliferation and IgG secretion P. falciparum malaria, as before mentioned, the role of APRIL in malaria is still unknown.In the recent years, several studies have been shown that BAFF, APRIL, and their receptors play a significant role in the pathogenesis of various noninfectious and infectious diseases P. vivax accounts for more than 85% of all malaria cases P. falciparum and P. vivax malaria in some adults from the Brazilian Amazon who were followed up during the acute and convalescent phases of infection.Brazil has a peculiar malaria epidemiological situation, in which P. vivax (n = 33) or P. falciparum (n = 19) malaria who resided in the communities of Porto Velho, the capital of the Rondônia state , in the Brazilian Amazon malaria endemic region . All malaria patients who were enrolled in this study complied with the following criteria: 1) they presented symptoms; 2) they had thin‐thick blood smears positive for malaria parasites; 3) they were not indigenous; 4) they were not prisoners; 5) they were all over 12 years old; and 6) no females were pregnant or breast‐feeding.P. vivax and/or P. falciparum were subsequently treated according to Brazilian Ministry of Health standards for malaria therapy: for uncomplicated malaria is recommended chloroquine combined to primaquine to P. vivax infection and artemether‐lumefantrine (Coartem®) to P. falciparum infection. Asexual blood forms of P. vivax and P. falciparum were cleared from the peripheral blood of all patients included in the study following therapy and no parasite reappearance was observed during follow‐up. The control group (n = 12) was composed of apparently healthy individuals who lived in the same geographic area, but were negative for malaria parasites as assessed by thick blood films and had not reported any malaria episodes for at least 5 years.The patients sought health care at the government outpatient clinic in Porto Velho after the onset of malaria symptoms. Blood samples were collected on the day of diagnosis before treatment (in the acute phase = D0) and 15 days later . After giving written informed consent which was reviewed and approved by the Ethical Committee of the Oswaldo Cruz Foundation (354/06), questionnaires were administered inquiring about demographic data, time of residence in the endemic area, personal and family malaria histories, presence of malaria symptoms, and use of malaria prophylaxis for each survey participant. After blood collection, donors positive for Plasmodium sp.) and species  were performed in all samples as previously described Venipuncture drew venous blood samples (5 mL) in EDTA‐containing tubes. Thin and thick blood smears of all donors were examined for malaria parasites. Parasitological evaluations were done by examination of 200 fields at 1,000‐fold magnification under oil‐immersion. The number of parasites was counted against 200 white blood cells (WBC). The parasite density per microliter of blood was calculated by multiplying the number of parasites counted by number of WBC divided by 200 7cells/mL in the freezing medium consisting of 90% heat‐inactivated fetal bovine serum  and 10% dimethyl sulfoxide  and placed into Nalgene cryogenic freezing container . The freezing container was then stored at −70°C overnight, and frozen cell samples were transferred to liquid nitrogen container (−196°C).WBC count was performed in the acute and during the convalescence phase using an automatic hematology analyzer (Pentra ABX). The cell counters provided data on leukocyte, lymphocyte, eosinophil, neutrophil, band cell, monocyte, and basophil counts. Qualified pathologists examined the smears. Of the subjects who agreed, venous peripheral blood (10 mL) was collected in heparin‐containnig tubes for flow cytometry analysis (five patients and four controls). Peripheral blood mononuclear cells (PBMC) were separated from whole blood by density gradient centrifugation . Cell viability was determined using the trypan blue dye exclusion assay. Cells were resuspended to a concentration of 2 × 10Cytokine and chemokine concentrations in plasma samples were determined by Luminex technology . Fourteen cytokines IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐10, IL‐12 p70, IL‐13, IL‐17, IFN‐γ, TNF‐α, G‐CSF, granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and three chemokines  were analyzed using a BioPlex‐Kit assay . The assay was performed according to the manufacturer's instructions using a BioPlex‐kit in combination with the Luminex system. Briefly, 50 μL of standard or test sample along with 50 μL of mixed beads were added into the wells of a pre‐wetted 96‐well microtitre plate. After 1 h of incubation and washing, 25 μL of detection antibody mixture was added and the samples were incubated for 30 min and then washed. Finally, 50 μL of streptavidin‐PE was added and after 10 min of incubation and washing, the beads were resuspended in 125 μL assay buffer and analyzed using a BioPlex suspension array system (Bio‐Rad Laboratories) and the Bio‐Plex manager software (v.3.0). A minimum of 100 beads per region were analyzed. A curve fit was applied to each standard curve according to the manufacturer's manual and sample concentrations were interpolated from the standard curves. The limit of cytokine detection using this method was 2 pg/mL for all cytokines and chemokines. The median cytokine and chemokine levels in 12 healthy controls were 2 pg/mL for IL‐2, IL‐4, IL‐5, IL‐6, IL‐7, IL‐10, IL‐12 p70, IL‐13, IL‐17, and GM‐CSF, 5.85 pg/mL for IL‐1β, 17.64 pg/mL for IFN‐γ, 23.23 pg/mL for TNF‐α, 4.40 pg/mL for G‐CSF, 379.13 pg/mL for MCP‐1, and 994.8 pg/mL for MIP‐1β.The plasma concentrations of APRIL and BAFF were measured with Human APRIL platinum ELISA Kit  and Human BAFF Instant ELISA kit  according to the manufacturer's instructions. Assays were performed in duplicate on 1/2 diluted samples. Plasma APRIL and BAFF levels were quantified using the in‐Kit standard curves for the respective cytokines. The minimal detectable doses of APRIL and BAFF in ELISA assay were 1.43 and 3.03 ng/mL, respectively. The median APRIL levels in 12 healthy controls were 5.02 ng/mL and the median BAFF were 6.72 ng/mL.Cryopreserved PBMC were thawed at 37°C, and Benzonase‐supplemented complete medium was slowly added  T test to calculate significance levels between patients and control group. Spearman‐rank correlation analysis was used to calculate correlations. Reported P values are two‐tailed, and statistical differences were considered significant when P values were less or equal to 0.05.The survey data were recorded and entered into a database created with EPI Info 2007 . Statistical analysis was carried out with GraphPad Prism 5.0  and R software. We previously checked whether the data was normally distributed by performing the D'Agostino's and Pearson omunibus normality test. As the data was not normally distributed we used Wilcoxon matched‐paired test to calculate significance levels between patients (D0 and D15) and Mann–Whitney unpaired P. vivax and P. falciparum malaria patient groups. Although the median of parasitemia appeared to be higher in P. vivax than in P. falciparum infected patients, this difference was not statistically significant. All patients were symptomatic at the time of enrollment independent of Plasmodium species and the most common symptoms reported by them were fever (91%), headache (89%), myalgia (77%), chills (77%), and vomiting (64%). However, no correlation was found between parasitaemia and axillary temperature (P < 0.05). There was no significant difference among the general features of the patient groups and control group, except for the absence of circulating parasites and the lowest number of previous malaria in the control group. All patients were parasitemia negative by day 15 of follow‐up after receiving effective drug treatment.General features of the volunteers are displayed in Table P. vivax (n = 33) or P. falciparum (n = 19). To investigate changes in APRIL and BAFF levels during the infection, we compared levels in the plasma from the same patient during the acute (D0) and convalescent (D15) phases.We analyzed APRIL and BAFF plasma levels from 12 healthy controls and 52 patients infected with P. vivax  and P. falciparum  than those of the controls . To investigate changes in APRIL levels during the infection, we compared APRIL levels in the plasma from the same patient during the acute (D0) and convalescent (D15) phases. During the convalescent phase P. vivax  and P. falciparum  patients had significantly lower levels of APRIL than during the acute phase. The levels of APRIL were similar between P. vivax and P. falciparum patients during both acute and convalescent phases .As shown in Figure P. falciparum patients  during the acute phase and returned to the levels similar to those of the controls . During the convalescent phase, P. falciparum  patients had significantly lower levels of BAFF than during the acute phase. In P. vivax patients, although the median BAFF levels were increased during the acute  and convalescent  phases, these levels were not statistically different from those of the controls . The levels of BAFF were similar between P. vivax and P. falciparum patients during both acute and convalescent phases . Furthermore, no correlation was found between APRIL and BAFF plasma levels during both acute and convalescent phases of P. vivax  and P. falciparum  malaria. The correlation between APRIL plasma levels and parasitaemia was only observed in P. falciparum malaria . There was no correlation between BAFF plasma levels and parasitaemia in P. falciparum  or P. vivax malaria .As shown in Figure P. vivax and P. falciparum‐infected patients  and convalescent (D15) phases. P. vivax and P. falciparum patients presented higher levels of IFN‐γ, TNF‐α, IL‐6, IL‐8, IL‐17, and MIP1β in the convalescent phase than in the acute phase. Although P. vivax and P. falciparum malaria patients have similar cytokine profiles during infection, P. falciparum patients presented higher levels of IFN‐γ and TNF‐α, in acute phase and of IFN‐γ, IL‐6, IL‐8, IL‐17, and G‐CSF in convalescent phase than P. vivax patients. Moreover, the levels of IL‐2, IL‐4, and IL‐12 were increased in the convalescent phase of P. falciparum infection. On the contrary, IL‐10 levels decreased in the P. vivax and P. falciparum malaria convalescent phase and returned to healthy normal levels. The cytokines IL‐5, IL‐7, and GM‐CSF were not detectable in most plasma samples. No differences were observed in MCP‐ 1 levels compared with controls. The lL‐10 and G‐CSF levels were positively correlated with parasitaemia in the P. vivax  and P. falciparum  malaria acute phase. The same was true for IFN‐γ , IL‐2  and IL‐4  in P. falciparum malaria patients.Table P. vivax acute phase, APRIL plasma levels were positively correlated with IL‐ 1 , IL‐2 , IL‐4 , IL‐6 , and IL‐13  while BAFF plasma levels were not statistically correlated with any of the cytokines/chemokines assayed. No correlation was observed between APRIL, BAFF, and cytokine plasma levels during the P. vivax convalescent phase. No relationship was found between any of the cytokines/chemokines assayed and the APRIL or BAFF plasma levels in P. falciparum acute or convalescent phases and control group .As shown in Table P. vivax malaria patients during the acute (D0) and convalescent (D15) phases and four healthy controls was performed  compared to the controls   .Transmembrane Activator and CAML Interactor (TACI) is a receptor of the APRIL/BAFF system and is expressed on B cells and activated T cells. To determine whether malaria induces increased expression of TACI, phenotypic analysis of PBMC of five ed Figs. A and 3B.Plasmodium infection Plasmodium immune‐modulatory molecules P. falciparum malaria, nothing is known about the role of the follows: 1) APRIL in any malaria or 2) BAFF/APRIL system in P. vivax malaria. In this regard, the main objective of this study was to investigate whether BAFF and APRIL plasma concentrations could be part of inflammatory response associated with uncomplicated P. falciparum and P. vivax malaria in some adults from the Brazilian Amazon that were followed up during the acute and convalescent phases of infection.Malaria is characterized by many pathophysiological changes including alterations in the immune system involving myeloid‐cells, T‐cells and B‐cells. Regarding B‐cells, a number of observations clearly demonstrate that B‐cell homeostasis is affected by P. vivax and P. falciparum infections whereas BAFF levels were only increased during acute P. falciparum malaria. These findings raise a possibility that the regulation of BAFF secretion may differ between the two strain infections. Although a range of myeloid cells produces BAFF and APRIL, the mechanisms regulating production and expression are poorly understood and likely differ from cell type to cell type. Plasmodium spp. infections elicit significant changes including immunological alterations involving myeloid cells and cytokine levels. In line with previous studies P. vivax and P. falciparum malaria  in malaria‐naive adults P. falciparum antigens (sPfAg) are capable of inducing BAFF release from monocytes and B‐cell co‐cultures P. falciparum and P. vivax are genetically distant malaria parasites P. vivax acute phase malaria, APRIL but not BAFF levels correlated positively with IL‐1, IL‐2, IL‐4, IL‐6, and IL‐13 levels. In addition, we did not find any relationship between P. vivax parasitaemia and APRIL levels, while an inverse correlation was found between P. falciparum parasitaemia and APRIL plasma levels. Although APRIL and BAFF share several biological characteristics and receptor‐specificity, they have distinct functions. While BAFF is fundamental for B‐cell maturation and survival, APRIL is essential for antibody class‐switching and plasma‐cell survival and is involved in the late stage of B‐cell differentiation First, we observed that the APRIL plasma levels were increased during acute ia Table . In bothConsistent with our previous findings P. vivax and P. falciparum malaria P. vivax and P. falciparum malaria.The APRIL and/or BAFF plasma levels are upregulated in many noninfectious P. vivax and P. falciparum acute phase malaria. However, lymphopenia was a transient finding and 15 days after the beginning of treatment, the lymphocyte counts were similar to those of control subjects, indicating that this period was sufficient for the patients to achieve lymphocyte homeostasis, according to our previous findings Consistent with previous findings P. falciparum malaria in Kenyan children. We have access to PBMC of five P. vivax malaria patients in acute and convalescence phases. Our results showed a transient and significant increase in the percentage of TACI‐positive CD4+ and CD8+ T cells during the acute phase, as well as a transient but not significant increase in the percentage of TACI‐positive CD19+ B cells in P. vivax infected donors. Aware that our sample size is too small to make inferences, we also can not rule out the possibility that the discrepancies observed between this study and ours with respect to TACI‐positive B cells, could be associated with the fact that patients involved in our study were adults with uncomplicated P. vivax malaria living in different epidemiological scenarios. In relation to T cells, TACI expression presented a transient kinetics similar to IL‐10 and APRIL levels. Thus, the APRIL and IL‐10 plasma levels as well as the proportion of CD4+ and CD8+ T cells expressing TACI might reflect the acute phase of P. vivax malaria, and may contribute to calcium‐dependent constitutive activation of nuclear factor of activated T cells (NF‐AT) TACI is a receptor of the APRIL/BAFF system and is predominantly expressed on B cells and in some subsets of activated T cells P. vivax infection. Moreover, it seems that the APRIL and BAFF inductions reflect different host strategies for controlling infection with different malaria species, P. vivax and P. falciparum. Further larger cohort study is ongoing in the Brazilian Amazon to provide better knowledge of the APRIL/BAFF system as well as the TACI B‐ and T‐cell expression in the immunomodulation of malaria.Taken together, we demonstrate that APRIL plasma levels as well as the percentage of TACI positive CD4+ and CD8+ T cells were increased in the acute phase of uncomplicated RAP performed experiments, statistical analysis and wrote the paper. ACS performed experiments and manuscript review. DSPS performed statistical analysis and manuscript review. RNRS performed experiments. LAS performed experiments. MRA performed statistical analysis. FS did fieldwork support. JCLJ performed collection of blood and epidemiological data and manuscript review. JOF performed collection of blood and epidemiological data. DMVV designed the study and performed manuscript review. PMDL performed the experiments, statistical analysis and manuscript review. CECP designed the study and performed manuscript review. DMB conceived, designed and coordinated the study, performed collection of blood and epidemiological data and wrote the paper. All the authors read and approved the manuscript for publication.None.


Seasonal malaria chemoprevention (SMC) for children < 5 is a strategy that is gaining popularity in West African countries. Although its efficacy to reduce malaria incidence has been demonstrated in trials, the effects of SMC implemented in routine program conditions, outside of experimental contexts, are unknown. In 2014 and 2015, a survey was conducted in 1,311 households located in Kaya District (Burkina Faso) where SMC had been recently introduced. All children < 72 months were tested for malaria and anemia. A pre–post study with control group was designed to measure SMC impact during high transmission season. A difference-in-differences approach was coupled in the analysis with propensity score weighting to control for observable and time-invariant nonobservable confounding factors. SMC reduced the parasitemia point and period prevalence by 3.3 and 24% points, respectively; this translated into protective effects of 51% and 62%. SMC also reduced the likelihood of having moderate to severe anemia by 32%, and history of recent fever by 46%. Self-reported coverage for children at the first cycle was 83%. The SMC program was successfully added to a package of interventions already in place. To our knowledge, with prevalence < 10% during the peak of the transmission season, this is the first time that malaria can be reported as hypo-endemic in a sub-Sahelian setting in Burkina Faso. SMC has great potential, and along with other interventions, it could contribute to approaching the threshold where elimination strategies will be envisioned in Burkina Faso. Its entire population lives in high-risk transmission areas. The 2015 World Malaria Report indicates that there were 5,428,000 confirmed cases and 17,000 deaths attributable to malaria out of a total population of 17 million, and the latest malaria indicator surveys revealed a parasitemia prevalence (detected by microscopy) of 46% in children 6–59 months.3 With 414 malaria cases per 1,000 persons at risk, Burkina Faso has one of the highest incidence rates in the world.4 Most of the malaria cases and deaths occur during or directly after the rainy season (approximately from July to October) and children < 5 are at the greatest risk; surveillance data indicate that they represented 51% of all malaria cases and 69% of all deaths attributable to malaria in 2016.9Despite encouraging signs of a recent reduction in transmission,11 Officially recommended by the WHO since 2012, SMC involves the administration of a long-acting antimalarial drug regimen that combines therapeutic and prophylactic effects.12 The regimen, commonly one single dose of sulfadoxine–pyrimethamine (SP) and three daily doses of amodiaquine (3AQ), is typically administered once per month to the entire population of children < 5 during malaria transmission season. SMC usually consists of three or four cycles of drug administration per year, depending on the length of the rainy season, and is most suitable in specific regions of the Sahel and sub-Sahel.13Because of high parasite prevalence and the highly seasonal nature of malaria transmission, most of the Burkinabe health districts are located in areas where the World Health Organization (WHO) has recommended seasonal malaria chemoprevention .15 Experimental studies have also found evidence of a lower prevalence of moderate to severe anemia, but this reduction was not always significant across trials.19 Similarly, some trials suggest that SMC is associated with greater weight gain or improvement of malnutrition indices.20 Monthly administration of SP-3AQ was found to be the drug regimen with the highest efficacy,23 and using community health workers (CHWs) to deliver it is more cost-effective than using facility-based nurses, immunization outreach clinics, or outreach trekking teams.25Clinical trials and meta-analyses have demonstrated the efficacy of SMC to reduce malaria incidence during the intervention period and parasitemia prevalence at the end of the transmission season and suggest a positive impact on all-cause mortality.27 This type of evaluation is challenging but necessary; indeed, the effectiveness of public health programs often differs from the efficacy measured in trials because of implementation challenges.28 In Burkina Faso, where SMC was recently introduced at the national level, a robust, quasi-experimental study was designed to evaluate its impact on malaria-related outcomes  in preschool-aged children. To the best of our knowledge, this was the first study to evaluate the impacts of an SMC program implemented under routine program implementation. As of 2017, 11 countries have adopted SMC  at different scales of implementation.29 The secondary objective was to assess self-reported SMC coverage among the targeted population and to identify the main factors associated with CHW visits and treatment administration during the first cycle of SMC.However, there is a lack of evidence on the protective effect of SMC in natural conditions of implementation outside of experimental contexts.31 The program is implemented in partnership with nongovernmental and international organizations that financially support the strategy, such as the Malaria Consortium, the World Bank, and United Nations International Children’s Emergency Fund.The National Malaria Control Program of Burkina Faso introduced SMC in 2014 in three health districts, then expanded to 17 districts in 2015 and 54 districts in 2016. The national policy builds upon previous trials and pilot projects conducted in the country.pfdhfr and pfdhps associated with SP resistance are rare,32 and is different from artemisinin-combination therapies (ACTs), the first-line treatment of malaria. CHWs administer SP and the first dose of AQ, and give the remaining two daily doses of AQ to the caregiver. Four cycles of SMC are conducted each year—once every 4 weeks during high malaria transmission season. SMC usually starts late July, but the exact starting date fluctuates every year depending on several factors, including logistical considerations and pluviometry.The recommended drug regimen for SMC is SP+3AQ. SP was selected because it is still highly effective for parasite clearance in Burkina Faso, where the gene mutations The target population for SMC comprises all children between 3 and 59 months, excluding those with known allergies to sulfamides or AQ, those under cotrimoxazole treatment, and those severely ill or experiencing a presumptive malaria episode. Children 3–11 months receive one 25-mg dose of sulphadoxine, one dose of 12.5 mg of pyrimethamine and three doses of 75 mg of AQ given over the course of three consecutive days. Children 12–59 months received double doses of SP and AQ. The children’s vaccination booklets are consulted to help determine their age.SMC is administered by CHWs. In teams of two, they go door-to-door to every household. They sometimes organize distribution sessions at gathering venues such as markets, churches, dwellings, mosques, and fields. After explaining SMC strategy , they administer treatments and refer children with malaria or danger signs to the closest health center. CHWs fill in forms where they indicate the number of treatments administered in every household, and they keep updated stock management sheets. Drugs are supplied through the Ministry of Health to the health centers, who then allocate them to the CHWs. Nurses in health centers are responsible for coordinating CHWs’ work and for collecting SMC forms. Supervisory visits are conducted by nurses and district health authorities. No supply issues were reported during the first cycle of SMC in 2015.33 The population study includes all children aged 3–71 months nested in 1,311 households. Observations from 2014 and 2015  were used for both the children exposed to SMC in 2015 and those who were not . The intervention (the first cycle of SMC) took place from July 31 through August 3, 2015. In 2014 and 2015, data collection started in mid-August and lasted about 3 weeks. No children were exposed to SMC in 2014. The investigators had no control over the implementation of the intervention. In particular, children were not assigned by the research team to the intervention or control group, the latter of which included all children not reached by the intervention at the time of the posttest survey in 2015. The second study component was aimed at measuring self-reported SMC coverage using posttest observations only.The impacts of SMC were evaluated through a quasi-experimental study, i.e., a pre–post with nonrandomized control group design.34 Kaya is located 100 km northeast of the capital, Ouagadougou. The climate is sub-Sahelian, with a long continuous dry season from November to June.35 In 2013, the total rainfall was 781 mm, 81% of which fell in 3 months–July, August, and September. There are six primary health centers in the study area, four rural and two urban, with a catchment population totaling ∼19,077 individuals.The study was carried out in two urban sectors of the city of Kaya  and in 15 villages located within a 20-km radius around the city. These sectors and villages were selected because they are part of the Kaya Health and Demographic Surveillance System (Kaya HDSS), which collaborates with the study. Further information about the Kaya HDSS is available elsewhere.9 Health care for children < 5 has been free of charge in Kaya since 2011, which increased the demand for services provided at health facilities for febrile children.36 Approximately 95% of malaria cases in the region are caused by Plasmodium falciparum,37 the principal vectors being Anopheles gambiae, Anopheles funestus, and Anopheles arabiensis.8The 2016 health statistics for Kaya district indicate that the test positivity rate for malaria is 77% in health facilities. Based on the number of cases routinely diagnosed, the average number of malaria infections per child per year is 1.1.6 The panel study was designed in 2011 to evaluate the effects of community case management of malaria on treatment-seeking practices and morbidity outcomes in children < 5 years. The panel originally consisted of 3,002 households selected in two health districts (Kaya and Zorgho). After 2013, funding was downsized, procedures and instruments were changed or simplified, and only households located in Kaya district were kept in the panel. The objective was to create a longitudinal research platform for evaluating natural experiments related to child health. More background information on the panel is available elsewhere.38 The population of the present study includes the 1,311 households surveyed in 2014–2015 which had at least one child aged 3–71 months. Although the targeted population for SMC comprises children aged 3–59 months, the age cutoff was extended to 71 months because targeting errors for SMC were anticipated for children between 5 and 6 years old. All households agreed to participate in the study.Households in the panel were randomly selected from the population living in the study area. They were all surveyed once a year during the peak malaria transmission season, late August or early September.lactate dehydrogenase (pLDH) malaria antigen; 2) period prevalence of parasitemia, defined as a positive RDT for the histidine-rich protein II (HRP2) malaria antigen; 3) prevalence of moderate to severe anemia, defined, according to the WHO, as a hemoglobin concentration under 10 g/dL39; and 4) a history of reported fever in the past 14 days. Whereas the pLDH antigen is cleared from the bloodstream within 3 to 5 days after an effective malaria treatment, the HRP2 antigen persists in the blood for 4 to 5 weeks after parasite clearance.43 The HRP2-based RDT consequently has a low specificity in posttreatment periods and should be considered a measure of period prevalence.46 Finally, the HRP2 antigen is expressed only by P. falciparum, whereas the pLDH antigen is expressed by any species of Plasmodium. A study conducted in Burkina Faso revealed that sensitivity was similar for detecting the two antigens by RDTs (99% for pLDH-based RDT and 100% for HRP2-based RDT), unlike their specificity .37The study has four outcome indicators: 1) point prevalence of parasitemia defined as a positive rapid diagnostic test (RDT) for the detection of parasite Two outcome measures were used for self-reported coverage assessment: 1) a household being visited by a CHW during the first cycle of SMC (household level) and 2) a child receiving a course of SP+3AQ . The information used to assess coverage came from the household survey in the panel.47 and Demographic and Health Surveys. Children’s characteristics  were recorded as well as self-reported information about exposure to SMC, presence of recent fever episodes, and malaria-related practices . Self-reported exposure to SMC was cross-validated by reviewing the children’s vaccination booklets, if any (only 60% of children had one). The household questionnaire explored the main socioeconomic characteristics and potential confounding variables: household location; size; distance between the household and the CHW; education and occupation of the parents; preventive practices and knowledge regarding malaria; possessions of the household; and finally water, sanitation, and hygienic practices of the household. Based on this self-reported information and direct observation of the households’ immediate environment , a socioeconomic status index and a hygiene index were created, as described elsewhere.48Questionnaires were all based on standardized instruments ® GPS device .Data collection was performed by teams of two: one field assistant and one health technician. Surveys were administered by trained field assistants using tablets. All surveyed households and CHW residences were located with a Garmin®, Suwon City, South Korea) was conducted using whole blood via finger prick to detect parasitemia. Results were read and interpreted according to the instructions provided by the manufacturer. If invalid, the RDT was performed a second time. Hemoglobin was measured using a photometer  and axillary temperature was recorded using a digital thermometer. Test results were entered into a data capture system and into record books as backup. If an eligible child was temporarily absent at the time of the survey, the fieldworkers went back to the household for a second and last time on the following day(s) to complete the tests. In the presence of a positive RDT, anemia and/or signs of malnutrition, the child was referred to the nearest health facility, and a referral note intended to the nurse was given to the parent—healthcare for children has been free of charge in Kaya District since 2011. Only one test was performed per child per year.After completing the questionnaire, all children (excluding neonates) were tested for malaria and anemia. A RDT .Three different types of analyses were performed: 1) coverage assessment, 2) impact evaluation, and 3) a placebo test as a sensitivity analysis for impact evaluation. All analyses were conducted using the STATA2 test was checked to assess goodness-of-fit. To ensure consistency between the analyses and comparability of the results, the same set of covariates was included in the different models, whatever the outcome or the type of analysis . Therefore, models are not as parsimonious as they could be, but their interpretation is more straightforward and coherent.SMC coverage was calculated among the study population by area type: urban, peri-urban, and rural. A multiple logistic model was fitted for the two binary outcomes and associations between these two outcomes, and the set of potential confounding variables (previously described) were investigated. Only postintervention data were used. For the first outcome (receiving the visit of a CHW), the level of analysis was the household, whereas for the second (being administered a course of SP+3AQ), it was the child. In the latter case, a random intercept for households was included in the model to account for the hierarchical structure of the data—children are nested in households. Multicollinearity was ruled out by using the Collin package (STATA Corp.) and verifying that variance inflation factors did not exceed 4. The statistical significance of the Wald χ49 First, the reduction in prevalence of morbidity attributable to the intervention was estimated. The causal effects of the intervention on the population under treatment (average treatment effect on the treated) was computed using a difference-in-differences approach.50 This approach is based on the comparison of change  in outcome between the intervention and the control group and has the advantage of controlling for time-invariant confounders.52 The second approach consisted of calculating the protective effects of the intervention for individuals. The protective effect of SMC is expressed by the regression coefficient for exposure, i.e., the adjusted risk ratio of having a positive outcome in the intervention group versus in the control group.Two approaches based on the Rubin–Neyman potential outcome framework were used to evaluate the impacts of SMC.55Average treatment effects on the treated and adjusted risk ratios are derived from the same generalized linear (Poisson distribution) model that was replicated for the four malaria-related outcomes. The Poisson regression with robust variance estimators was preferred over the logistic distribution to obtain model-based risk ratio estimates, as recommended in the literature on impact assessment.58 To produce the propensity scores, a multiple logistic model was fitted to estimate the adjusted probabilities of selection into the treatment and control groups, which were then used to generate scores to be included in the causal models.58 All variables potentially related to the outcome were included in the propensity score, regardless of whether they were related to exposure.60 The balance of covariates was checked by testing the weighted differences in the means of every covariate between the groups.58 No difference was found to be statistically significant at a threshold of 10%.The causal models were fitted with a propensity score weighting to control for possible selection bias on the set of potential confounding variables (previously described).Random intercepts for children were included to account for the hierarchical structure of the data . Multicollinearity was ruled out using the same procedures as described previously. Goodness-of-fit was assessed by computing the Pearson-based dispersion statistics; they suggested the absence of residuals’ over-dispersion and indicated that the data fitted reasonably well the models. The Akaike information criterion and the Bayesian information criterion were considered when comparing models with and without propensity score weighting.62 Material and procedures for data collection in the panel were identical between 2013 and 2015.Difference-in-differences approaches assume a common trend between the treatment and control group . To assess the robustness of this assumption, we conducted an antitest (or placebo test) verifying whether variations in outcomes were equivalent between the two groups during 2 years of pre-intervention (2013 and 2014).Ethical approval was obtained from the research ethics committee of the University of Montreal Hospital Research Center in Canada and Burkina Faso’s health research committee. Data were used in conformity with Kaya Health and Demographic Surveillance System policy (authorization 1KH2016-02). Written informed consent was obtained every year from every respondent and from the caretakers of children on their behalf. Children with danger signs were immediately referred to a health center.N = 2,291) were present at the time of the survey: 1,820 (79%) had received an SMC treatment (intervention group), 462 (20%) had not received an SMC treatment (control group), and 9 (0.4%) did not remember or refused to answer. Population characteristics are summarized in 2 < 0.01; P value = 0.947) with RDT results the previous year (2014), indicating no selection issues on the likelihood to receive SMC based on the outcome. Differences were found in certain characteristics between children who received SMC in 2015 and those who did not. As expected, more ineligible children (aged 60–71 months) were found in the control group than in the intervention group . Children in the intervention group were more likely to live in polygamous households than their counterparts in the control group . Finally, the mean hygiene score was higher in the intervention group than in the control group .In 2015, there were 2,523 children aged 3–71 months in the panel. Among these, 91% .Estimates derived from regression models with and without propensity score weighting are displayed in Supplemental File 1).For each of the four malaria-related outcomes, Supplemental File 2).The protective effect of SMC for individuals is expressed by the risk ratio of having a positive outcome in the intervention group versus in the control group. At baseline, the likelihood of having a positive pLDH-based RDT in children who received SMC the next year was not different than in children who did not receive SMC in 2015  and 62% , respectively. SMC also reduced the likelihood of having moderate to severe anemia and recent history of fever by 32%  and by 46% , respectively. All these estimated protective effects are in the anticipated direction .Supplemental File 3). Therefore, there is no indication that the assumption of similar trends between the two groups is not plausible.There was no statistical difference in changes between intervention and control groups in pre-intervention years (2013–2014) for any of the outcomes  positive impact on child morbidity in the study area. The point prevalence of malaria was reduced by 3.3% points (95% CI = [−0.08 to 0.01]), which translated into a protective effect (adjusted risk ratio) of 51% (95% CI = [0.24–0.99]). The protective effect on the period prevalence was larger, 62% (95% CI = [0.29–0.52]). This is unexpected because HRP2-based RDTs take more time to turn negative after parasite clearance than pLDH-based RDTs, and therefore are less sensitive to measure SMC therapeutic effect between two cycles.45 A plausible explanation is that a floor effect occurred, as the overall point prevalence of malaria was already low in the study area. This may have contributed to making the evaluation of impacts less precise and reducing the power of statistical tests, as shown by the large 95% CI = for this particular outcome.It has been estimated that ∼20,000 deaths and 5 million cases of malaria could be averted with SMC in the Sahel and sub-Sahel regions, if this intervention were widely deployed.16 This was anticipated and is understandable given the study context. Indeed, impacts were assessed in our study under routine program conditions. Taking that into consideration, results are of critical importance for public health planning; decision makers can anticipate that SMC reduces malaria prevalence by at least 50% in a targeted population.The protective effects on malaria infection observed here are lower than the 85% efficacy reported in the only trial that used the same regimen and measured prevalence during the SMC round rather than after.63 The protective effect for this outcome was 32%. Similar to results observed from trials, the treatment effect for anemia was lower than that for malaria, which is understandable as only a fraction of the risk of anemia is attributable to malaria.17 That being said, reducing the risk of anemia by a third through an SMC campaign is a tremendous gain in terms of public health, knowing the heavy burden caused by this condition in children under age 5.64SMC reduced the prevalence of moderate to severe anemia by 16% points (average treatment effect on the treated). This corroborates previous experiments showing that malaria chemoprevention or intermittent treatment is most likely to reduce anemia in rural areas.56 Results from both types of models were presented for the reader to appreciate and compare them. Estimates with and without propensity scores are similar for the malaria parasitemia outcomes, suggesting that selection bias is unlikely.65 However, they differ moderately for anemia and history of fever. Imbalances between the two groups, either random or systematic, seem to have more influence for these two outcomes. One hypothesis is that, because of a more complex etiology, the association between exposure and these outcomes is more affected by covariates than the association between SMC and malaria parasitemia outcomes.Models with propensity score weighting have the potential to better adjust for covariates and, in this analysis, presented signs of better goodness-of-fit  but at the expense of larger standard errors for the estimates.24 used the same delivery method for SMC and found that coverage for children was 100% at the first cycle, then gradually decreased to 69% at the fourth cycle. If a similar drop in coverage was to occur in the study area, the proportion of children fully covered at the end of the round would be 57%. Several factors could explain the limited coverage for households and for children, notably their absence during the rainy season. Indeed, it is common for some household members to spend all day in the fields. Some even leave their homes and temporarily live in shelters closer to the fields.66 Also, it is common for young children to stay with their mother during the day and accompany her wherever she goes. This could explain why, even if a household was visited, some children did not receive SMC treatment. Acceptability was likely not an issue because no parent reported having refused SMC for their child. However, troubles with drug administration (difficulties in crushing the tablets into powder and the presence of side effects) could have affected adherence.Local contingencies may have reduced the fidelity of SMC during the first year of its implementation and, consequently, limited its effectiveness in the study area. Self-reported coverage for the first cycle of the 2015 round was 76% and 83% at the household and individual levels (children < 5 years), respectively. Patouillard et al.48 At the same time, results show that households are not more prone to be visited by a CHW if they have a higher hygiene score, indicating no selection bias from the CHW based on the households’ hygiene score. Although it has been demonstrated that domestic environmental measures have a direct preventive impact on malaria,67 further investigation is required to interpret the positive association between a household hygiene score and the uptake of an intervention against malaria such as SMC.None of the observable variables influenced the heterogeneity in coverage among households, and only two variables were associated with the likelihood of receiving SMC: the child’s age and the hygiene score of the household. The first association was expected, if only because age is an eligibility criteria for SMC and because older children are less often at home. The second association with hygiene score suggests that children are more prone to receive SMC if they live in a household whose immediate surroundings are cleaner , even after adjusting for the area and the household socioeconomic status.70 Although better integration of these efforts has been recommended,71 SMC contributed positively to this package.The repeated annual surveys also indicate that malaria transmission in the study area is now moderate. Even among children who did not receive SMC, malaria point prevalence was only 6.5%, whereas previous surveys in the panel reported a prevalence of 18.6% (2013) and 15.0% (2014). The overall reduction in malaria prevalence in 2015 might be partly due to the fact that a package of interventions against malaria has been introduced since 2010 in the district, including adoption of ACTs as first-line treatments, community case management, repeated campaigns of universal bed net distribution, community sensitization, and removal of user fees at health centers.73 With all these interventions targeting children < 5, the age pattern of the malaria burden may shift to school-aged children, as shown in other studies.75 The extension of SMC to school-aged children may be an option to further reduce malaria transmission and approach the threshold where elimination strategies will be envisioned.77To our knowledge, this is the first time that malaria has been reported as hypo-endemic in a sub-Sahelian setting in Burkina Faso.80 Second, diverse analytical methods were combined to control for observable and time-invariant nonobservable confounding factors: multiple models, propensity score weighting, and difference-in-differences approach. Third, analyses were intentionally conservative; bilateral statistical tests were used, and children were included in the analysis even if they were recently sick and/or received an ACT treatment. Finally, post hoc analyses were conducted to test the hypothesis of similar trends during pre-intervention period and strengthen the robustness of difference-in-differences estimates.Several measures were taken to increase the internal validity of the outcome evaluation in this quasi-experimental study. First, the study was built on a robust design (pre–post with control group), and the conclusions were fueled by four outcome indicators, covering various components of the chain of effects .82 However, preliminary results from a qualitative study on SMC implementation show a good fidelity to the plan and acceptability of the population, which is congruent with the positive effects on morbidity measured here. The possibility of a selection bias affecting the results is small, although the risk cannot be completely ignored in any quasi-experimental study, no matter how robust.33The sensitivity analyses and placebo tests (antitests) do not suggest the presence of selection biases or residual confounding. Coverage assessment has not indicated self-selection issues in treatment administration. It was not possible to assess the impact depending on the adherence level, because implausibly high values were self-reported, as seen in other studies.77 It was impossible to investigate this hypothesis here because only the postintervention measure was taken a few weeks after the first SMC cycle.Finally, the power of the study has been limited by an unexpectedly low malaria transmission rate in the study area. Despite this, significant clinical and statistical gains were observed. The high level of replicability in this study confirms our appreciation of the internal validity and capacity to attribute the gains to SMC. The external validity is limited by the fact that the study took place in only one district. Variations in the implementation of the strategy between districts in Burkina Faso, and between Sahelian countries, are possible. It seems important to replicate this study elsewhere and measure how sensitive the results are to variations in context and in implementation fidelity. In theory, repeated cycles of SMC could contribute to reduce overall malaria transmission.During the first year of its introduction under routine program conditions, SMC already had large and positive impacts for the targeted population. The prevalence of all malaria-related outcomes under study  decreased immediately after the first cycle of the SMC campaign. This translated into large and significant protective effects for the children who received the chemoprevention treatment. SMC was successfully added to the package of interventions already in place in the health district where the study was conducted, even if some implementation barriers, notably limited coverage, likely reduced its effectiveness. To our knowledge, it is the first time that malaria can be described as hypo-endemic in the area, with a parasitemia prevalence < 10% during the peak of its transmission season. SMC is an intervention with great potential in Burkina Faso, and along with other interventions, it could contribute to approaching the threshold where elimination strategies will be envisioned.Supplemental Files.


This study aims at investigating the relationship between malaria and blood group types in east China. Between 1 January 2011 and 31 March 2017, 99 malaria patients were enrolled for the study. Laboratory tests were conducted on their infection status and blood types. Clinical data of the participants were retrieved for analysis. Plasmodium falciparum, 8 (8.08%) were infected with Plasmodium vivax, and only 1 (1.01%) was infected with Plasmodium malariae. The most common blood group among the participants was group O (38.38%) followed by blood groups A, B, and AB, with 32.32%, 22.22%, and 7.07% cases, respectively. There was no significant relationship between the prevalence of malaria and ABO blood types (P > 0.05). In the blood group O, the prevalence of haemolytic-uremic syndrome and cerebral malaria was 13.16% and 5.25%, respectively, which was lower than that of the other three blood types (P > 0.05). There was no mortality during the period of study. Overall, 90 (90.91%) of the patients were positive for There was no mortality among the malaria patients in this study. The blood group O was the most common blood type. Due to small sample size of data, there was no significant association between ABO blood types and malaria infection. Malaria is a life-threatening parasitic disease transmitted by mosquitoes. Positive prevention and treatment for malaria have resulted in a wide-scale reduction in incidence and mortality; however, 198 million cases (uncertainty range: 124–283 million) and 584,000 deaths  still occur yearly [The relationship between ABO blood types and malaria susceptibility has been studied by several researchers; however, the results have been contradictory. Bayoumi et al.  observedAmong the global malaria burden, 90% has been estimated to occur in Sub-Saharan Africa ; hence, This retrospective study was conducted in the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Department of Infectious Diseases, the First Affiliated Hospital, College of Medicine, Zhejiang University, China. The research was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University, which waived the need for consent because the study was retrospective and the data were analysed anonymously.Between 1 January 2011 and 31 March 2017, malaria patients aged over 18 years were admitted into the First Affiliated Hospital, College of Medicine, Zhejiang University, China. All patients were hospitalized due to fever. Patients with positive bacterial blood cultures or those with obvious bacterial, viral, or other parasitic infections were excluded. For the control group, we randomly selected 100 subjects from those that presented with fever but were negative for malaria. Of the control group, patients with complicated underlying diseases were excluded. The demographic and clinical details of the malaria patients were obtained from the case records of the patients.Two reviewers at a reference parasitology laboratory were blinded of the thin and thick blood smears and independently confirmed the malaria diagnoses. The blood groups were determined using the forward and reverse method. Haematological parameters, which included haemoglobin, total leukocyte count, and platelet count of each patient, were done on an automated cell counter.P value < 0.05 was considered statistically significant for all analyses.The statistical analysis was performed using SPSS version 18.0 . Chi-square test was conducted to calculate and compare the categorical variables. The measurement data between different ABO blood types were compared with single-factor analysis of variance. In this study, 99 malaria patients (41.82 ± 14.68 years) were enrolled, with a male preponderance (90.91%). All the 99 malaria patients were treated and discharged from the hospital or had improved and were discharged with drugs. No mortality was recorded during the period of study. One hundred patients  who were hospitalized due to fever without malaria were randomly selected as control group. Plasmodium falciparum, 8 (8.08%) were infected with Plasmodium vivax, and only 1 (1.01%) was infected with Plasmodium malariae. Moreover, 97 (97.98%) patients were imported malaria cases. Before being diagnosed with malaria, 94 patients worked or travelled in Africa and 3 patients travelled in Southeast Asia.Of the 99 malaria patients, 90 (90.91%) were infected withDuring treatment, 75 patients received artemisinin and 24 patients received dihydroartemisinin piperaquine phosphate tablets. Pyronaridine phosphate, chloroquine, and primaquine were used in 6, 4, and 11 patients, respectively.The most common blood group among the 99 malaria patients was group O (38.38%) followed by blood groups A (32.32%), B (22.22%), and AB (7.07%) respectively. The distribution of ABO blood types among the control group was A (32%), B (23%), O (32%), and AB (13%) respectively. There were no differences in the distribution of ABO blood group types among the malaria patients and control group . MoreoveThe relationship between ABO blood types and malaria was first suggested over 50 years ago , and newAmong the malaria patients, blood group O had the highest prevalence with 38.38%. This result agrees with previous studies, which reported a high frequency of blood group O in tropical regions with rampant malaria , 11, 12. Plasmodium falciparum infection [Among malaria patients with blood group O, the prevalence of haemolytic-uremic syndrome and cerebral malaria was 13.16% and 5.25%, respectively, which was lower than that of the other three blood types. Therefore, we considered blood group O to be prone to less severe malaria, although no statistically significant relation was found. Toure et al.  and Hegdnfection , 12, 15.Our study has some limitations. First, the small numbers of malaria patients and control group may lead to statistical errors. Second, malaria patients in this study were concentrated in the Zhejiang Province; the data may be insufficient to evaluate the relationship between ABO blood types and malaria in east China. Future studies with an increased scale are warranted in more regions to explore whether the result is similar in other populations.In conclusion, there was no mortality among the malaria patients in this study. There was no significant association between ABO blood types and malaria infection. However, the blood group O is the most common blood type in malaria patients in east China, who are prone to less severe malaria. Therefore, malaria patients with blood group O seemingly have mild clinical outcomes.


Plasmodium in India is key to improving malaria surveillance and diagnostic tools. Here we describe serological profiling of immune responses at three sites in India by probing protein microarrays consisting of 515 Plasmodium vivax and 500 Plasmodium falciparum proteins with 353 plasma samples. A total of 236 malaria-positive (symptomatic and asymptomatic) plasma samples and 117 malaria-negative samples were collected at three field sites in Raurkela, Nadiad, and Chennai. Indian samples showed significant seroreactivity to 265 P. vivax and 373 P. falciparum antigens, but overall seroreactivity to P. vivax antigens was lower compared to P. falciparum antigens. We identified the most immunogenic antigens of both Plasmodium species that were recognized at all three sites in India, as well as P. falciparum antigens that were associated with asymptomatic malaria. This is the first genome-scale analysis of serological responses to the two major species of malaria parasite in India. The range of immune responses characterized in different endemic settings argues for targeted surveillance approaches tailored to the diverse epidemiology of malaria across the world.Understanding naturally acquired immune responses to  Plasmodium vivax and Plasmodium falciparum are its main causes. While the two major malaria parasite species co-occur in India, their proportion varies across the country. Antibodies in an individual indicate current or past Plasmodium infection, and can be used to identify suitable vaccine candidates, as well as develop novel tools for malaria surveillance. We present the results of a pilot study undertaking the first large-scale characterization of antibody responses to ~1000 Plasmodium antigens at three field sites in India using high-throughput protein microarray technology. Individuals from the eco-epidemiologically diverse sites showed reactivity to 265 P. vivax and 373 P. falciparum antigens, regardless of infection status. Further comparison of individuals with symptomatic and asymptomatic malaria revealed the most immunogenic Plasmodium antigens, as well as antigens that were recognized with greater intensity in individuals that were asymptomatic at the point of sample collection. These results are a valuable addition to existing data from other malaria endemic regions, and will help to expand our understanding of host immunity against the disease.Although malaria deaths have fallen by 60% worldwide since 2000, the disease remains a significant public health problem. India has the highest burden of malaria in the South-East Asia Region, where  The burden of malaria in India has halved over the last 15 years, yet India continues to account for over 70% of malaria cases in South East Asia . Long-laPlasmodium is multi-faceted, involving both the humoral and cell-mediated response pathways. CD8+ effector T cells can kill intra-hepatocytic stages [Plasmodium was first demonstrated by passive transfer of antibodies from a clinically immune adult to a symptomatic child, which conferred protection from severe disease [P. falciparum, and more recently for P. vivax, in regions of both high and low transmission [P. vivax infection than to P. falciparum [P. vivax to maintain a dormant state within hepatocytes [P. vivax and P. falciparum [The human immune response to the malaria parasite c stages , while mc stages , 7. The  disease . Antibod disease , 11. Dessmission , 13. Intlciparum , which, atocytes . Additiolciparum –19.Plasmodium parasites, current infections, or past infections, and can therefore be used to identify suitable candidates for vaccine development, and to develop tools that can estimate malaria transmission levels [e.g., immunofluorescent antibody test, enzyme-linked immunosorbent assay etc., or that detect antibodies to several antigens simultaneously, such as genome-scale protein microarrays [Plasmodium among Indian populations have been sparse [P. vivax and ~500 P. falciparum antigens, with sera from individuals at three sites in India–Raurkela in the eastern state of Odisha, Nadiad in the north-west state of Gujarat, and Chennai in the southeastern state of Tamil Nadu. Although the two major malaria parasite species co-occur at all three sites, they differ in their prevalence. Historically, P. vivax has been predominant in Chennai and Nadiad, whereas P. falciparum has been predominant in Raurkela. The objectives of our study include: 1) to provide a descriptive analysis of seroreactivity profiles of individuals against Plasmodium antigens at three epidemiologically diverse malaria endemic sites in India; 2) to examine the relationship between age and the acquisition of antibodies against Plasmodium antigens; 3) to identify the most immunogenic Plasmodium antigens based on antibody responses at three sites in India; and 4) to identify antigens recognized with greater intensity by individuals with asymptomatic malaria.Antibodies in an individual can be indicative of recent exposure to n levels  or monitn levels . Antibodroarrays . Previoun sparse , with onn sparse –26. Protn sparse ; howeverEthical approval to conduct this study was obtained from New York University Institutional Review Board (Study #i10-00173) and the Ethics Committee of the National Institute of Malaria Research, India. All project staff completed Protection of Human Research Subjects training prior to beginning the study, and clinical samples were collected after informed consent was obtained from all participants. For all adult patients, informed written consent was obtained from literate patients, and oral consent (documented by a thumb print) was obtained from illiterate patients. For child participants, assent was obtained from the participant, in addition to written or oral consent from their parent or legal guardian.Samples were collected as part of epidemiology studies at three sentinel sites in India:  Chennai P. vivax is the dominant species, and although transmission is perennial, malaria cases peak between July and October. Subjects were enrolled at the Besant Nagar Malaria Clinic, or in cross-sectional surveys conducted in the Besant Nagar catchment area, which is composed of middle- and upper- class urban dwellings, a few slums, and a large coastal fishing community.Chennai is the largest city in the southern state of Tamil Nadu, with a population of over 7 million in 2011. Located on the eastern coast of India, it has a tropical wet and dry climate, with a rainy season focused between mid-October to mid-December. Chennai accounts for 55.6% of all malaria cases in Tamil Nadu, and had an annual parasite index (API) of 1.79 in 2013 . EntomolP. vivax and P. falciparum species, with slightly higher prevalence of P. vivax, and an average API of 2.5 (range 0.87–4.12) and EIR of 0.05–0.21 [Nadiad, with a population of 0.2 million in 2011 is located in the Kheda district in Gujarat state. Rainfall is primarily received between June and September. Nadiad is hypo-endemic for both .05–0.21 . The NatP. falciparum is the dominant species in Raurkela, and it has the highest EIR (7.3–127) and API  of the three field sites in our study [Raurkela, with a population of over 0.5 million, is located in Sundargarh district close to the northern border of the state of Odisha, and has a tropical climate, with high temperatures and heavy rainfall between June-September and December-January. ur study . SubjectPlasmodium [Sample collection and processing at these sites has been described in detail elsewhere –31. To sasmodium , and pla. A total of 236 individuals were diagnosed as malaria-positive and 117 individuals were diagnosed as malaria-negative by microscopy and PCR. Individuals ≤ 15 years were categorized as children, and those > 15 years were categorized as adults. Of the 236 malaria-positive individuals diagnosed by PCR, 147 individuals with documented fever (body temperature ≥ 37.5°C) or a history of fever in the past 48 hours were categorized as symptomatic; 89 individuals without documented fever or history of fever in the past 48 hours were categorized as asymptomatic. Pooled plasma samples from 20 healthy semi-immune adults from Sepik, Papua New Guinea, collected in 2004 and where transmission of P. falciparum and P. vivax was equally high, were used as positive controls, and pooled plasma samples from unexposed individuals in the United States were used as negative controls.Plasma samples from 353 individuals that were randomly selected from clinic and cross sectional studies at our field sites were utilized for this study  reactions. Accession numbers and description of the antigens are based on the P. vivax Salvador I and P. falciparum 3D7 genome annotation from the PlasmoDB database [The Pf/Pv500 protein array  comprises a total of 515 database . Completdatabase .2 transformed to generate median-normalized fold-over-control (FOC) values. To generate the heat map, median-subtracted intensity values were obtained by subtracting the median intensity of IVTT controls from the raw intensity of IVTT protein spots. Significance Analysis for Microarrays [2(FOC) ≥ 1. The breadth of antibody response was determined as the number of antigens an individual or group of plasma samples were seropositive to, based on the above criteria. Comparison of the breadth of responses across various groups was carried out using the Kruskal Wallis test with p-values adjusted using Dunn’s correction for multiple testing. Analysis of differential antibody reactivity between groups was performed by a two-sample T test assuming unequal variance and corrected for false discovery rates using the Benjamini-Hochberg method. Statistical analyses were carried out using Microsoft Excel, R v3.0.1, and Prism v7.0. All data have been made publicly available through PlasmoDB [Microarray spot intensities were quantified using the ScanArray Express software . The non-specific background signal for each spot was calculated as the median intensity of sample-specific no-DNA IVTT control spots. For data normalization, the raw intensity values for IVTT proteins were divided by the median intensity of corresponding IVTT controls and logroarrays  was carrPlasmoDB .P. vivax and 500 P. falciparum antigens to evaluate the seroreactivity of 236 malaria-positive and 117 malaria-negative individuals  from three study sites, Raurkela (n = 168), Nadiad (n = 114) and Chennai (n = 71), in India. Table 1 provides characteristics of the individuals and study sites.We used the Pf/Pv500 protein microarray containing 515 P. vivax and 373 P. falciparum antigens. The global profile of antibody binding to Plasmodium antigens at the three sites is shown . The overall seropositivity or breadth of response against P. falciparum antigens was higher compared to P. vivax antigens.Comparison of antibody responses between all plasma samples identified 638 antigens recognized with significantly higher intensity in Indian samples versus unexposed U.S. controls. All subsequent analyses were carried out using this subset of 265 P. vivax and P. falciparum antigens in adults. Malaria-positive adults from Nadiad showed significantly higher breadth of response to P. vivax antigens compared to malaria-positive adults from Raurkela (p < 0.03) and Chennai (p < 0.0001). Malaria-negative adults from Nadiad also showed higher levels (p < 0.03) of seropositivity to P. vivax than adults from Chennai . In the case of P. falciparum antigens, malaria-positive adults from Chennai had significantly lower seroreactivity compared to malaria-positive adults from Raurkela (p < 0.0001) and Nadiad (p < 0.002); a similar observation was made upon comparison between malaria-negative adults from Chennai with Raurkela (p < 0.002) and Nadiad .To analyze observed differences in seroreactivity across the three Indian sites in more detail, we compared the breadth of response to P. vivax  and P. falciparum , as well as greater intensity of antibody binding against P. vivax  and P. falciparum  when compared to children.We compared the breadth and intensity of antibody response between malaria-positive children (age ≤ 15 years) and adults (age > 15 years) to understand age-based differences in acquired immunity (limited to Raurkela and Nadiad due to insufficient numbers of children sampled in Chennai). Adults showed higher breadth of response against Plasmodium proteins across all study sites. For every antigen, we calculated the average reactivity and breadth of response in malaria-positive and malaria-negative samples at each site. We identified 26 Plasmodium antigens with average log2(FOC) ≥ 1 among malaria-positive samples at all sites; these antigens were also recognized by most of the malaria-negative samples at each site. This list of 14 P. vivax and 12 P. falciparum antigens represents the most immunogenic Plasmodium antigens that are indicative of current or past malaria infections . In the case of P. vivax, merozoite surface proteins, ETRAMP, SFT2, and a number of hypothetical proteins are recognized with the greatest intensity. Among the P. falciparum antigens recognized with the greatest intensity of antibody binding are members of the PfEMP1 family, PTP5, AMA1, and HSP70.We identified highly immunogenic Plasmodium-positive individuals were further categorized as symptomatic or asymptomatic. We compared the breadth and intensity of antibody response to Plasmodium antigens in adults classified into these two categories. Of the 174 malaria-positive samples, 121 were diagnosed by both PCR and microscopy, and 50 were detected only by PCR, i.e., were submicroscopic. Analysis of the 121 microscopy- and PCR-positive adults revealed that asymptomatic adults infected with P. falciparum showed significantly higher breadth  and intensity  of antibody response than symptomatic adults. In addition, we observed that the asexual parasitemia levels in asymptomatic adults were much lower  compared with symptomatic adults infected with P. falciparum. In the case of P. vivax, no significant differences in the breadth  and intensity .Based upon the presence or absence of fever at the time of enrollment and up to 48 hours earlier, 2(FOC) values to identify antigens that were differentially reactive between asymptomatic and symptomatic adults . Asymptomatic adults infected with P. falciparum displayed significantly higher reactivity to several P. falciparum antigens compared to symptomatic adults. We have identified 19 P. falciparum antigens that were recognized with 2-fold greater intensity in asymptomatic adults in India (Table 3). On the other hand, no P. vivax antigens showed significantly higher reactivity in asymptomatic compared to symptomatic individuals infected with P. vivax.We were interested in identifying antigens that have significantly higher reactivity in sera from asymptomatic individuals compared to symptomatic individuals. We used logPlasmodium antigens in the Indian population using protein microarray technology. The three field sites in our study are distributed across eco-epidemiologically diverse regions in India, with differing prevalence of P. vivax and P. falciparum. We observed a broad response to P. vivax and P. falciparum antigens at all three sites, regardless of malaria infection status. Overall levels of antibody binding were greater in P. falciparum antigens compared to P. vivax antigens, as previously reported in regions with co-endemic occurrence of P. vivax and P. falciparum [P. vivax and 373 P. falciparum antigens compared to unexposed US controls.We present the first genome-scale analysis of seroreactivity to lciparum . Our IndP. falciparum, we observed that decreasing prevalence corresponded with a decrease in the breadth of antibody response. Malaria-negative adults showed a similar pattern to malaria-positive adults at all sites, reflecting the background seroreactivity due to parasite exposure. The breadth of response to P. falciparum antigens was significantly lower in adults from Chennai compared to both Raurkela and Nadiad; although the mean breadth of response appeared to be lower in Nadiad compared to Raurkela, the decrease was not statistically significant.In the case of P. vivax. Malaria-positive adults from Nadiad showed significantly larger breadth of response than adults from both Chennai and Raurkela; this was not observed in malaria-negative adults. We were puzzled that regardless of their malaria infection status, the seroreactivity of individuals from Chennai was similar to Raurkela, which has much lower prevalence of P. vivax. Since we began the study in 2012, the incidence of malaria has gone down in India, particularly in the state of Tamil Nadu where Chennai accounts for ~55% of the malaria burden [P. vivax and P. falciparum in Raurkela and Nadiad may have been higher than expected during the course of our study. Hypothetically, P. vivax seroreactivity may also be affected by differential rates of P. vivax relapse; sites with a higher relapse rate may exhibit greater priming of the immune response. However, we do not have data on relapse rates at our three sites to address this conclusively. Additionally, the breadth of response could be affected by differences in the total number of individuals and the proportion of symptomatic and asymptomatic infections between the sites.We did not observe a correlation between the breadth of antibody response and species prevalence for a burden . Thus, ta burden . TherefoP. vivax as well as P. falciparum antigens was significantly higher in adults than in children.The breadth and magnitude of immune response have been known to increase with age, as a consequence of repeated exposure to the parasite. We compared the responses of children and adults from Nadiad and Raurkela to determine the influence of age on the immune response against malaria. The breadth and intensity of response against Plasmodium parasite species varies across India, we were interested in identifying highly immunogenic antigens from all three sites that could be used to develop a serological assay for countrywide routine surveillance. It is also important to elucidate the kinetics of antibody acquisition and maintenance [P. vivax proteins in the Indian population, such as ETRAMP, MSP10, MSP8, and hypothetical proteins such as PVX_117680, PVX_083560, PVX_097730, PVX_110935, PVX_118705 have also been identified as highly immunogenic in other studies [P. falciparum identified in our study, members of the PfEMP1 family, PTP5, MSP10 and HSP70 were also identified as immunogenic or recognized with greater intensity in asymptomatic malaria in other ICEMR studies [Antigens recognized with the highest intensity by sera from both symptomatic and asymptomatic malaria patients may serve as indicators of exposure. Data from other ICEMR studies  have higntenance  in order studies –42. Amon studies , 43, 44. studies .P. falciparum infection have lower average asexual parasitemia, but higher breadth as well as intensity of response, than adults with symptomatic infection. Asymptomatic P. falciparum infection was also associated with significantly higher seroreactivity to several P. falciparum antigens as compared to symptomatic infection, and these antigens may serve as novel vaccine candidates in addition to the limited repertoire of candidates currently being developed. A majority of the antigens associated with clinical immunity in P. falciparum infections are either exported to the infected red blood cell during the intraerythrocytic stages of parasite development  or present on the merozoite surface . These proteins are exposed to the host immune system for the longest duration of the infection, facilitating the development of a strong immune response. Some of these antigens play essential roles in vital processes such as invasion (RON2), making them promising vaccine candidates. GCN5 and LSA3, previously associated with protection from experimental challenge with sporozoites [Asymptomatic individuals may not be protected from malaria parasite infection, but they may possess immunity against symptomatic disease. Our data indicate that adults with asymptomatic rozoites , 47, androzoites , 35, 43 P. falciparum is associated with a broad and intense response to several antigens, but with low parasitemia levels. In contrast, adults with asymptomatic and symptomatic P. vivax infection had comparable breadth and intensity of antibody response, as well as similar asexual parasitemias. In addition, asymptomatic P. vivax infection was not associated with higher seroreactivity to specific P. vivax antigens. Based on these findings, it appears that unlike P. falciparum, antibody-mediated immune responses may have a much lower contribution in asymptomatic P. vivax infections. P. vivax is thought to be more pyrogenic than P. falciparum, since it stimulates a stronger fever-inducing cytokine response despite frequently presenting at a lower parasite burden than P. falciparum [et. al., propose that regulatory cytokines, such as IL-10 may play a more critical role in protecting P. vivax patients from severe clinical complications, while a strong inflammatory response may be critical in controlling parasite density in P. falciparum infections [Comparison of serological profiles of adults with symptomatic or asymptomatic malaria suggests that immunity to lciparum . Howeverfections .Plasmodium antigens, and provide a means to identify highly immunogenic proteins and antigens recognized more strongly in asymptomatic individuals, which may subsequently be used for the development of vaccines and routine surveillance tools. We acknowledge the limitations of using this array, since the antigens have been produced from sequence information of decades-old single reference isolates . Our future studies will include country-specific redesign of the arrays using sequence data from diverse parasite strains circulating at our study sites, in order to capture regional diversity in antigenic genes. In addition, appropriate study design will be extremely important to tease out the complexities of the host immune response to malaria. In particular, prospective cohort studies, with careful monitoring of parasite transmission and patient serology, are likely to be the most informative. In conclusion, these data have broadened our understanding of naturally acquired immunity against P. vivax and P. falciparum in India, and will contribute to global malaria control and eradication measures.Our pilot study was conducted in tandem with other protein microarray projects within the ICEMR program and using the same Pf/Pv500 protein array , 35, 43.


Levels of both angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) correlate with malaria disease severity and are proposed as biomarkers and possible therapeutic targets. To establish their role in malaria, a systematic review was performed of the literature on Ang-1 and Ang-2 with regard to their potential as biomarkers in malaria and discuss their possible place in adjuvant treatment regimens.Ten electronic databases were systematically searched to identify studies investigating Ang-1 and Ang-2 in human and murine malaria in both clinical and experimental settings. Information about the predictive value of Ang-1 and Ang-2 for disease severity and their regulatory changes in interventional studies were extracted.Plasmodium falciparum patients to study their ability as biomarkers could distinguish between multiple disease severity states; the more the disease severity states differed, the better they could be distinguished. Five studies differentiating malaria survivors from non-survivors with Ang-2 as marker found an AUROC in a range of 0.71–0.83, which performed as well or better than lactate. Prophylactic administration of FTY720, rosiglitazone or inhalation of nitric oxide (NO) during malaria disease in mice resulted in an increase in Ang-1, a decrease in Ang-2 and an increased survival. For rosiglitazone, a decrease in Ang-2/Ang-1 ratio was observed after post-infection treatment in mice and humans with malaria, but for inhalation of NO, an effect on Ang-1 and survival was only observed in mice.Some 579 studies were screened; 26 were included for analysis. In all five studies that determined Ang-1 levels and in all 11 studies that determined Ang-2 in different disease severity states in falciparum malaria, a decline in Ang-1 and an increase of Ang-2 levels was associated with increasing disease severity. All nine studies that determined angiopoietin levels in Both Ang-1 and Ang-2 levels correlate with and can distinguish between malaria disease severity states within the group of malaria-infected patients. However, distinct comparisons of disease severity states were made in distinct studies and not all distinctions made had clinical relevance. Changes in levels of Ang-1 and Ang-2 might also reflect treatment effectiveness and are promising therapeutic targets as part of multi-targeted therapy.The online version of this article (doi:10.1186/s12936-016-1624-8) contains supplementary material, which is available to authorized users. Plasmodium falciparum, one of the six Plasmodium species  that can infect humans. The high pathogenicity of P. falciparum is in part related to the expression of P. falciparum erythrocyte membrane protein-1 (PfEMP-1) on the membranes of infected erythrocytes. The infected erythrocytes with PfEMP-1 on the membrane adhere to the vascular endothelium of vital organs. This process is called sequestration and will cause a partial obstruction of the blood flow. Together with the increased deformability of uninfected erythrocytes and adherence of uninfected erythrocytes to infected erythrocytes, which is called rosetting [P. vivax, which may run a relatively benign course compared to P. falciparum, is most important and can also cause severe malaria and result in death. The pathogenesis of P. vivax infection differs from P. falciparum infection and is still poorly understood. Plasmodium vivax infects only young erythrocytes (reticulocytes) which can also adhere to the vascular endothelium, but showed ten times less cyto-adhesion compared to P. falciparum-infected erythrocytes in vitro [P. vivax showed a stronger response than P. falciparum [P. vivax leads to disproportional organ-specific parasitaemia and what role the cytokine response plays in the pathogenesis remains unclear.Even though many disease control interventions have been implemented, malaria remains a major health problem with an estimated 214 million cases and 438,000 deaths worldwide in 2015 [P. falciparum infection as PfEMP-1 on the erythrocyte membranes interacts with several cyto-adherence receptors that show a low, basal expression pattern in a non-inflammatory environment, but are upregulated during malaria infection [Endothelial cell activation is crucial in the pathogenesis of nfection . Activatnfection , fibrin nfection , 8, compnfection , or platnfection , endothenfection , 11. Endnfection  and dengnfection , 14. To nfection –20). TheAng-1 and Ang-2 are ligands of the Tie-2 receptor, which is expressed on endothelial cells and regulates endothelial quiescence during normal physiological conditions. Ang-1 is constitutively produced and excreted into the blood by pericytes and smooth muscle cells and it is also stored in platelets. Ang-1 binds to the Tie-2 receptor, thereby acting as an agonist. Upon Ang-1/Tie-2 interaction, the most important downstream signalling event is protein kinase B phosphorylation by phosphoinositide-3-kinase resulting in an anti-apoptotic and anti-inflammatory status of the endothelial cell.Ang-2 is produced in endothelial cells and pre-stored in the Weibel–Palade bodies (WPB) together with vWF. Upon activation of endothelial cells, exocytosis of the WPB is induced and their content is released into the blood stream . The incDuring malaria, a decrease in Ang-1 and an increase in Ang-2 concentration have been observed . It is nBecause of the correlation of Ang-1 and Ang-2 levels with disease severity and mortality, Ang-1 and Ang-2 have been evaluated as biomarkers for malaria disease severity in multiple studies. However, each individual study compared different disease severity states and the studies were performed in distinct populations at different geographical locations around the world. To gain more insight in their role during malaria, the current literature concerning the use of Ang-1 and Ang-2 as biomarker in a clinical setting is here reviewed systematically. Furthermore, the role of Ang-1 and Ang-2 as potential therapeutic targets in malaria is evaluated as they might play a causative role in the pathogenesis.Plasmodium. In Embase, Medline and CInahl, thesaurus terms were used together with words in title and/or abstract. In the other databases only titles and/or abstracts were searched. The databases Embase.com, Medline (Ovid), Cochrane Central registry of trials, Web of Science, Scopus, cinahl (EBSCOhost), Pubmed as supplied by publisher , lilacs, scielo, ProQuest, and Google scholar , were searched without data limit, from inception lastly on 15 June 2016  studies performed in non-pregnant humans; and, (II) interventional studies. The aim of studies in I was to correlate Ang-1 and Ang-2 levels with disease severity and in II to find an adjuvant therapy next to anti-malarial treatment.Plasmodium species, the study population, the Ang-1 and/or Ang-2 protein and/or mRNA levels or the Ang-2/Ang-1 ratio in various disease states, and information about the associations of Ang-1 and Ang-2 levels with other variables. In the studies included in II, data were extracted regarding study subject, mice species or population characteristics, details of the studied intervention, Ang-1, Ang-2 protein or mRNA levels and the Ang-2/Ang-1 ratio.For the articles of I, information was extracted on the Plasmodium infection were identified and included for analysis; 22 studies were performed in humans only, of which five included children and adults in Asia and Canada as a single group , 0.02; ii: 0.79 (0.72–0.84), 0.03) [Since cerebral malaria is a complication of falciparum malaria and therefore a manifestation of severe malaria, distinguishing cerebral malaria from other forms of severe malaria was difficult, as shown by the observation that only the Ang-1 concentration had a predictive value significantly higher than chance  with a s), 0.03) . The com), 0.03) .Plasmodium species, the Ang-1 and Ang-2 concentration may also be used for distinguishing cerebral malaria from other diseases involving the central nervous system (CNS) in critically ill patients. Distinguishing cerebral malaria from other febrile diseases affecting the CNS is challenging, but can have major treatment implications. Ang-1 was found to differentiate between cerebral malaria, diagnosed by malaria specific retinopathy, and other febrile CNS-affecting diseases with a sensitivity and specificity of 88% (95% CI 69–96) and 87% (95% CI 73–94), respectively -infected mice and/or P. falciparum-infected humans received next to artesunate either a functional S1P receptor antagonist , production of the Ang-1 levels was 30–50% compared to wild type mice five to six days post-infection [del mice treated with rosiglitazone [When rosiglitazone was given to nfection . An incrlitazone . This emiNO as adjuvant therapy for severe malaria and rosiglitazone for uncomplicated malaria was also studied in humans. In a total of 272 Ugandan children, adjuvant iNO was given in two studies with either nitrogen  or air  as placeThe role of Ang-1 and Ang-2 as biomarkers and possible therapeutic targets in severe malaria was evaluated in this systematic review. Decreased Ang-1 and increased Ang-2 concentrations seem to be valuable as biomarker for disease severity and survival. The role of Ang-1 and Ang-2 in follow-up of treatment effectiveness, as well as their role as future therapeutic targets, are promising, but further studies are warranted. Nonetheless, some discussion points before use as biomarkers or therapeutic targets remain.P. falciparum with coma , or malaria with coma persisting >30 min after a seizure). Nevertheless, Ang-1, Ang-2 and the Ang-2/Ang-1 ratio could be valuable markers if they can identify patients with uncomplicated malaria at risk for progressing to severe disease. The area under the receiver operating characteristic (AUROC) for distinguishing uncomplicated malaria from severe malaria is 0.88 (p = 0.001) and the sensitivity and specificity are 86% (95% CI 71–94) and 85% (95% CI 77–92), respectively [The results indicate that the decreased Ang-1 and the increased Ang-2/Ang-1 ratio are robust biomarkers to distinguish uncomplicated malaria from cerebral malaria , 29, 32.ectively   pathogen-related factors, such as infections with different strains with inherently differences in pathogenicity; and, (3) environment-related factors, such as differences in access to healthcare that may result in delayed diagnosis or delayed start of proper treatment. In addition, the methods by which Ang-1 and Ang-2 levels are determined are further developing and currently lack proper standardization. Additional investigations are required before conclusions on the varying levels can be drawn.Even though Ang-1 and Ang-2 concentrations could discriminate between disease severity states during malaria, distinct studies found varying levels of sensitivity and specificity . Ang-1 tivation . In addi limited . These diewed by ). More iPlasmodium species infections possibly plays a role in the lack of effect of adjuvant treatment when given post infection. During malaria disease in humans and mice the immune system, coagulation system and the activity of endothelial cell are all systematically altered. These processes are complex and interweaved with each other (see Fig. The complexity of the host response to see Fig. . Providisee Fig. , althougAng-1 and Ang-2 concentrations correlate with disease severity and survival in malaria and show a good discriminative power to distinguish disease severity states with predictive values equal to, or higher than, the currently used biomarkers plasma lactate and parasitaemia. However, different disease severity states were compared in the distinct studies and not every distinction has clinical impact. The possible ability of Ang-1 and Ang-2 concentrations to distinguish cerebral malaria from other febrile CNS-affecting diseases and to predict those patients progressing from uncomplicated malaria towards severe malaria will have clinical value and should be studied in more detail before it can be used in routine practice.For further optimization of treatment of patients with severe disease, multi-targeted adjuvant treatment regimens are needed. Agents interfering with Ang-1 and or Ang-2 may be included in such adjuvant therapy as their role as therapeutic target is promising. However, it is conceivable that agents interfering with multiple pathways are necessary, since multiple regulatory systems are consecutively activated during malaria infection.