Malaria, anaemia and malnutrition are frequently co-existing diseases that cause significant morbidity and mortality particularly among children. This study measured the prevalence, intensity and evaluated risk factors for malaria parasitaemia, anaemia and malnutrition among children living at low versus high altitude settings in the Mount Cameroon area.A cross-sectional community based survey involving 828 children aged 6 months to 14 years was conducted between July and November 2017. Malaria parasitaemia was confirmed by light microscopy, haemoglobin concentration was measured using an auto haematology analyser, nutritional status was determined from the anthropometric measurements collected, and socioeconomic status related variables by the use of questionnaire. Anaemia and malnutrition were defined according to World Health Organization standards. Associations between predictor variables and primary outcomes were assessed using logistic regression analysis.Malaria parasite and anaemia were prevalent in 41.7% and 56.2% of the children, respectively while, malnutrition prevalence was 34.8% with wasting, underweight and stunting occurring in 25.7%, 19.9% and 23.7% of them respectively. Overall malaria parasite geometric mean density was 413/µL of blood . The odds of having malaria parasitaemia was highest in children 5–9 years of age , living in lowland  as well as those whose domestic water was collected from an open source (streams/springs)  than their counterparts. Being < 5 years  or 5–9 years  of age, having malaria parasite  and fever in the past 2 days  were identified as significant risk factors of anaemia while the age group < 5 years was the only significant risk  associated with malnutrition.While age specific attention should be given in the control of malaria (5–9 years), anaemia (< 10 years) and malnutrition (< 5 years), the existing malaria control programmes should be revised to integrate anaemia and malnutrition control strategies so as to improve upon the health of the children. The proportion of the population at risk in sub-Saharan Africa who are infected with malaria parasites is estimated to have declined from 22% in 2005 to 13% in 2015, leading to a decrease, from 146 million to 114 million  and highlands (> 600 m asl). The coordinates of Batoke ranged from altitude 8 m, latitude 04°01.364′N, longitude 009°05.971′E to 47 m, 04°02.039′N and009°05.808′E. Tole is located between 627 m, latitude 04°07.057′N, longitude 009°15.178′E and 630 m, latitude 04°6.906′N, longitude 09°14.434′E. The Mount Cameroon area stretches from the Atlantic Ocean at the Gulf of Guinea, gradually increasing from Limbe to 800–1200 meters in Buea on the eastern flank of the Mountain. The temperatures in lowlands (Limbe) are high and fairly constant ranging between 25 and 30 °C on average . The Tol species .The study was carried out among pre and school age children of both sexes aged 6 months to 14 years old, weighed > 5 kg, free from other clinical conditions not related to malaria and sickle test negative. Children with severe malaria  were excluded from the study.2pq/d2 [This cross sectional community based study was carried out between the months of July and November 2017, reported as the peak malaria transmission period in the Mount Cameroon area . Ensuing2pq/d2  where n The axillary temperature was measured using a digital thermometer and fever was defined as temperature ≥ 37.5 °C . AnthropA semi-structured questionnaire was administered to the child’s parent/caregiver to collect data on (i) demographics ; (ii) Socioeconomic status related variables   and knowPlasmodium species were observed on the blood film. The number of parasite were counted per 200 leukocytes on thick blood film and asexual parasite densities per μL of blood was obtained by multiplying the parasite count with the participants’ white blood cell count obtained from the complete blood count analysis. If gametocytes were seen, the count was extended to 500 leukocytes. Parasitaemia was categorized as low (< 1000 parasites/μL blood), moderate (1000–4999 parasites/μL blood), high , and hyperparasitemia  [Four millilitres of venous blood samples were collected from the children using sterile disposable syringes. The collected blood was aliquot into labelled ethylenediaminetetraacetate (EDTA) tubes and the remaining blood dispensed on slides for the preparation of thick and thin blood films. Labelled blood samples in EDTA tubes were transported on ice in a cool box to the Malaria Research Laboratory, University of Buea for a full blood count analysis. Thin blood films were fixed with absolute methanol and later stained along with thick blood films using 10% Giemsa for 20 min. The films were examined following standard procedure for the detection and identification of malaria parasites . As a qu,000 μL) .P. falciparum parasitaemia (of any density) and Hb < 11 g/dL. Non-malarial anaemia was defined as children with anaemia and without a malaria-positive smear for P. falciparum.Haematological parameters were assessed using an auto-haematology analyser (MINRAY 2800 BC), following the manufacturer’s instructions. A complete blood count was obtained and anaemia was defined as Hb < 11.0 g/dL  and furt2). Group means were compared using analysis of variance (ANOVA), Student’s t test and non-parametric tests such Mann-Withney U test and Kruskal–Wallis test where appropriate. Parasite density was log transformed before analysis. Associations between predictor variables and primary outcomes were assessed using both bivariate and multivariate logistic regression analysis. Multi-collinearity test was performed for all potentially correlated variables and only variables with variance inflation factors less 2 were included in the models. Odd ratios (ORs) and 95% confidence intervals (CIs) were computed. Any covariate with a P value < 0.2 in the bivariate analysis was subsequently included in the final multivariable logistic model. Significant levels were measured at 95% CI with the level of significance set at P < 0.05.Data collected was cleaned up and analysed using the IBM-Statistical Package for Social Sciences (IBM-SPSS) version 20 and Epi-info version 7. Continuous variables were summarized into means and standard deviations and categorical variables reported as frequencies and percentages were used to evaluate the descriptive statistics. The differences in proportions were evaluated using Pearson’s Chi Square . Fever, malaria parasite, anaemia and malnutrition were observed in 9.5% (79), 41.7% (345), 56.2% (465) and 34.8% of the children, respectively.The prevalence of falciparum malaria among the 828 children varied with altitude. Children of the lowlands had a significantly higher (P = 0.004) prevalence of malaria parasite (46.7%) than those of the highlands (36.9%), as shown in Table As shown in Fig. Plasmodium infection when compared with those whose domestic water was collected from a close source (tap and borehole).A logistic regression model demonstrated altitude (P = 0.008), age groups (P = 0.006) and water source (P = 0.005) as significant predictors of malaria parasite prevalence as shown in Table The prevalence of anaemia was significantly higher (P < 0.001) in the youngest age group (66.1%) than those older and in children who were malaria parasite positive 66.4%) than those negative as shown in Table % than thRelating to anaemia severity, children in highland had a higher prevalence of severe (2.5%) and moderate anaemia (14.8%) than children in lowland  and the difference was statistically significant at P = 0.005.As shown in Table As shown on Fig. The logistic regression model with anaemia status as dependent variable and altitude, age, gender, level of education and marital status of guardian, nutritional status, malaria status as well as fever history as the independent variables revealed the age group (P = < 0.001) malaria status (P = < 0.001) and fever in the past 2 days (P < 0.04) as significant risk factors of anaemia as shown in Table As shown in Fig. The prevalence of malnutrition varied significantly (P < 0.001) with the age group. The highest prevalence was observed in children of the < 5 years age group (55.7%) and lowest in children 5–9 years old (21.1%). Similarly, the prevalence of stunting 35.6%), underweight (32.9%) and wasting (29.0%) was higher in the under-five age group compared to the older children  and children of the < 5 years age group (P < 0.001) were significantly at odds of being malnourished. However, in the final multivariable model, age was the only significant predictor where children under 5 years of age were three times more likely to be malnourished than those older as shown in Table P. falciparum malaria, anaemia, and malnutrition as public health problems in children < 15 years across low and highland altitudes in the Mount Cameroon area. The overall malaria parasitaemia of 41.7% observed by microscopy in the study population reveals malaria remains a major cause of illness during childhood. The observation is similar to that reported by Lehman et al. [This cross-sectional study examines n et al.  in schoon et al.  and the n et al. . Even thn et al.  in the Mn et al. , the decPlasmodium to complete its sporogonic cycle within a shorter time in the mosquito vector [In line with Kimbi et al.  and Ndamo vector .Findings from the study revealed the odds of having malaria was highest in the 5–9 years age group. The epidemiological shift in malaria burden from the under-five age group to the 5–9 years age group is in line with previous surveys in this part of the country , 40. ThiIn line with previous study in Rwanda , living The high prevalence of overall anaemia in children less than 15 years highlights the impact of anaemia among the population in this area. The relationship between malaria parasitaemia and anaemia is well established in previous studies , 12, 37.Interestingly, in this study, children with fever for the past 2 days were 1.52 times more likely to have anaemia. A study carried by Sumbele et al.  in the sA lower haemoglobin level, but not anaemia, was significantly associated with malnutrition and particularly stunting in the study. Evidence for the impact of under-nutrition on development of anaemia in young children living in malaria-endemic areas had been reported previously . AlthougMalnutrition was common (34.8%) in the community with an overall prevalence of 23.7% for stunting, the most common form of malnutrition. The prevalence of stunting was lower than the 42.9% obtained by Akiyama et al.  in Loa PIn line with previous findings , 44, theThe study had as limitations some unmeasured factors such as micronutrient deficiency and markers of inflammation which may have acted as confounders on the risk of the presence of anaemia. Never the less, the findings of the study demonstrated the main factors associated with the presence of the public health problems of malaria parasitaemia, anaemia and malnutrition.While malaria, anaemia and malnutrition are still of public health concern in the Mount Cameroon area, there is a strong association between malaria and anaemia but not malnutrition. Even with the decline in morbidity (GMPD), the prevalence of falciparum malaria is highest among the 5–9 years age group when compared with the under-five or 10–14 years age group. Therefore, in the strategic planning and design of malaria control programmes due attention should be given to children residing in lowland, those whose domestic water is sourced from an open source and children in the age group 5–9 years. For the control of anaemia consideration should be directed to the younger age group (< 5 and 5–9 years), those with malaria parasitaemia, and children with a history of fever within the past 2 days. Despite the fact that stunting is the most common form of malnutrition, with males being more malnourished than females the insidious impact of stunting resulting to lower haemoglobin levels than in healthy children warrants thoughtfulness in planning its control. Hence the health of children in malaria meso-endemic areas can be improved upon if the existing malaria control programmes are revised to integrate anaemia and malnutrition control strategies.


In view of recent evidence from a randomized trial in Burkina Faso that periconceptional iron supplementation substantially increases risk of spontaneous preterm birth (< 37 weeks) in first pregnancies , explanation is required to understand potential mechanisms, including progesterone mediated responses, linking long-term iron supplementation, malaria and gestational age.The analysis developed a model based on a dual hit inflammatory mechanism arising from simultaneous malaria and gut infections, supported in part by published trial results. This model is developed to understand mechanisms linking iron supplementation, malaria and gestational age. Background literature substantiates synergistic inflammatory effects of these infections where trial data is unavailable. A path modelling exercise assessed direct and indirect paths influencing preterm birth and gestation length.A dual hit hypothesis incorporates two main pathways for pro-inflammatory mechanisms, which in this model, interact to increase hepcidin expression. Trial data showed preterm birth was positively associated with C-reactive protein (P = 0.0038) an inflammatory biomarker. The malaria pathway upregulates C-reactive protein and serum hepcidin, thereby reducing iron absorption. The enteric pathway results from unabsorbed gut iron, which induces microbiome changes and pathogenic gut infections, initiating pro-inflammatory events with lipopolysaccharide expression. Data from the trial suggest that raised hepcidin concentration is a mediating catalyst, being inversely associated with shorter gestational age at delivery (P = 0.002) and positively with preterm incidence (P = 0.007). A segmented regression model identified a change-point consisting of two segments before and after a sharp rise in hepcidin concentration. This showed a post change hepcidin elevation in women with increasing C-reactive protein values in late gestation . Path modelling confirmed seasonal malaria effects on preterm birth, with mediation through C-reactive protein and (non-linear) hepcidin induction.Following long-term iron supplementation, dual inflammatory pathways that mediate hepcidin expression and culminate in progesterone withdrawal may account for the reduction in gestational age observed in first pregnancies in this area of high malaria exposure. If correct, this model strongly suggests that in such areas, effective infection control is required prior to iron supplementation to avoid increasing preterm births.Trial registration NCT01210040. Registered with Clinicaltrials.gov on 27th September 2010 In 2014, estimates for prevalence of PTB in sub-Saharan Africa based on a variety of assessment methods ranged from 8.6 to 16.7% , very siHepcidin is an iron-regulatory hormone produced in the liver that controls the entry of iron into the circulation and tissue iron distribution . HepcidiPlasmodium falciparum parasites sequester in gut epithelium secondary effects on intestinal cell integrity, cell signalling and permeability are considered.In the PALUFER trial malaria prevalence was high above 50% . The triPlasmodium parasitaemia prevalence by microscopy was 54.3%, at ANC1, and prevalence did not differ by trial arm. Free treatment was available for women with fever or other malaria symptoms, but most trial participants were asymptomatic . Prevalence of placental malaria parasites at delivery was 33%. In women remaining non-pregnant parasitaemia prevalence was 41% at end assessment after 18 months weekly iron supplementation. Iron-supplemented non-pregnant women also received more antibiotic treatments for non-genital infections (P = 0.014); mainly gastrointestinal infections (P = 0.005), anti-fungal treatments for genital infections (P = 0.014) and analgesics (P = 0.008), than controls.The PALUFER trial has previously been described in detail  to better prepare girls for their first pregnancy . WhetherBackground and published data on the PALUFER trial, its design and results , 13–15, Literature was searched for references illustrative of plausible mechanisms operating for each stage of the model pathways leading to PTB in a malaria endemic area. Search terms focussed on markers of inflammation that could affect birth outcomes. Assumptions for an inflammation-driven model for iron induced excess spontaneous preterm births in the PALUFER trial were formulated derived primarily from published trial results.Relationships between PTB and biomarkers were assessed using logistic regression models, plotting fitted probabilities with associated 95% CI. The relationship between maternal serum hepcidin and CRP concentrations was strongly non-linear and was fitted using a segmented linear regression model which fits a continuous line composed of straight-line segments with a change of slope at a fitted change point. More complex models were considered including trial arm as a covariate, but as there were no substantive effects of supplementation, these were not more informative and the univariate analyses are presented. Biomarker levels between PTB and term birth groups were compared using Mann–Whitney U-tests. A path model was constructed based on the formulated schematic which incorporated C-reactive protein (CRP) and hepcidin mediation pathways and PTB. Season was used a proxy for malaria infection. The model was fitted to the PALUFER data as described in Additional file The trial results showed associations between maternal serum CRP concentrations > 5 mg/l at the first  and second  scheduled antenatal visits  and spontaneous PTB incidence (< 37 weeks gestation) . At ANC2In normal pregnancy hepcidin expression decreases physiologically in the second and third trimesters, thereby increasing the supply of iron to the circulation . As malaP. falciparum infection. Such changes may cause increased intestinal microbiota [Plasmodium parasites can have an impact on composition of gut microbiota although bi-directional mechanisms are not well understood, especially in pregnancy [Pathological changes (including detachment of epithelia and shortening of colonic villi) occur in the permeability, leakage of infected erythrocytes into the lumen and dysbiosis of the intestinal gut during crobiota . The extcrobiota , 19 and crobiota  and bactcrobiota  pathogencrobiota , 23 and crobiota . Malariacrobiota , which ccrobiota , 27. In regnancy . Reactivregnancy , 29. NO Clostridium perfringens, may influence the level of lipopolysaccharide binding protein and CRP in blood plasma [Figure d plasma  Strong ad plasma . Placentd plasma . TLRs ard plasma .Salmonella. This is because iron-loaded macrophages, which increase with hepcidin expression, have an impaired potential to kill various pathogens. This is partly attributed to reduced formation of NO which is essential to fight infection, as iron blocks transcription of inducible NO synthase (iNOS) [Hepcidin expression is reported to negatively affect proliferation of intrahepatic sporozoites , but cone (iNOS) . RegulatP. falciparum in the placenta and parasite adherence to gut endothelial cells with altered intestinal barrier function [P. falciparum infections. Malaria in pregnancy is characterized by the accumulation of infected red blood cells, leukocyte infiltration, and excessive fibrin deposition in the intervillous space of the placenta [Concurrent effects of pregnancy malaria are shown in Fig. function , 39. Adhplacenta , and is placenta . Placentplacenta , with inplacenta . Oxidatiplacenta . Placentplacenta , 45, regplacenta  and NLRPplacenta . HaemozoThe induction of inflammatory cytokines can modulate pregnancy outcomes with beneficial and/or detrimental effects . MaintenSalmonella typhimurium during malaria may facilitate bacterial colonization [S. typhimurium [S. typhimurium benefits from iron availability during malaria infection [S. typhimurium. Evidence is required to substantiate this association in young adults and pregnant women.The inflammasome promotes maturation and secretion of pro-inflammatory cytokines IL-1β and IL- 18, which results in pro-inflammatory cell death . Nod-Liknization , and exphimurium . In vivonfection . Higher nfection , may favP. falciparum infection prevalence [A maternal cortisol response occurs with inflammation. In non-endemic malaria areas a majority of studies have reported a consistently negative association between maternal cortisol and infant birthweight –64. In cevalence , 66. Theevalence , associaevalence . Inflammevalence . Progestevalence , with fuevalence , 71, or evalence . Hepcidievalence . Young mevalence .The characteristics of pregnancy malaria and enteric infection outlined in Fig. A path model was constructed based on the schematic in Fig. There was no statistically convincing evidence for any other mechanistic paths beyond the model presented.P. falciparum prevalence is high, especially in adolescents and primigravidae [The conditions for this PTB model presuppose a setting in which inflammation is due to a common exposure which is experienced by most women, such that the gestational age distribution is shifted to the left with the resultant gestational effect unlikely to be specific to a sub-phenotype . In othegravidae . A populgravidae . In suchgravidae . With higravidae .The inflammatory stimulus defined by CRP was associated with higher hepcidin concentrations with increasing CRP levels in both non-pregnant women and in primigravidae at ANC1. Hepcidin elevation occurred at lower CRP levels (< 5 mg/l) later in gestation at ANC2 Fig. , which wBody iron concentration was higher in women with malaria in this cohort of women . Women wP. falciparum infection [A limitation of this analysis is the scarcity of data for comparative analysis and lack of data on enteric biomarkers or helminthic infections from the PALUFER trial, although all participants received single doses of albendazole and praziquantel at enrolment. Data on serum or red cell folate was not available to assess whether folate status was an additional factor in the model. For example, all women had received 2.8 mg weekly folic acid, a dose which may provide sufficient substrates to enhance folate metabolism in nfection , therebyOther field trials assessing periconceptional nutrient interventions have mostly been undertaken in non-malaria endemic areas , and effInterventions to prevent PTB focus mainly on managing risk factors . ImproviAdditional file 1. Background to the PALUFER safety trial of periconceptional iron supplementation. This specifies key published trial results referring to references [ferences , 13–15.Additional file 2. Laboratory methods. Summary of laboratory methods used for CRP, hepcidin assays and malaria microscopy.Additional file 3. Gestational age distribution in days of livebirths in iron and control arms. Vertical stippled lines indicate 43 weeks and 37 weeks gestation.Additional file 4. Path modelling of PTB and gestation. Outline of Path Model, statistical methodology and results. Figures S1. (Path Model); Figure S2. (the fitted relationship between CRP and hepcidin); Table S1. (Path model coefficients).


Plasmodium infections in endemic areas are often asymptomatic, can be caused by different species and contribute significantly to transmission. We performed a cross-sectional study in February/March 2016 including 840 individuals ≥ 1 year living in rural Gabon (Ngounié and Moyen-Ogooué). Plasmodium parasitemia was measured by high-sensitive, real-time quantitative PCR. In a randomly chosen subset of P. falciparum infections, gametocyte carriage and prevalence of chloroquine-resistant genotypes were analysed. 618/834 (74%) individuals were positive for Plasmodium 18S-rRNA gene amplification, of these 553 (66.3%) carried P. falciparum, 193 (23%) P. malariae, 74 (8.9%) P. ovale curtisi and 38 (4.6%) P.ovale wallikeri. Non-falciparum infections mostly presented as mixed infections. P. malariae monoinfected individuals were significantly older (median age: 60 years) than coinfected (20 years) or P. falciparum monoinfected individuals (23 years). P. falciparum gametocyte carriage was confirmed in 109/223 (48.9%) individuals, prevalence of chloroquine-resistant genotypes was high , including four infections with a new SVMNK genotype. In rural Gabon, Plasmodium infections with all endemic species are frequent, emphasizing that malaria control efforts shall cover asymptomatic infections also including non-falciparum infections when aiming for eradication. Despite this, between 2014 and 2016 there was a considerable increase in number of malaria cases, showing that it needs continuous efforts to have sustainable success. To reach the ambitious aim of eradication, control measures have to remain constantly high also including asymptomatic and submicroscopic carriers to eliminate this constant reservoir. Malaria control efforts should additionally include remote areas, where maintaining health care is difficult. This investigation analyzed by molecular methods the silent reservoir of malaria parasites in a rural area of Gabon, Central Africa.Due to huge efforts in elimination and eradication of malaria the numbers of malaria cases and deaths reduced substantially according to WHO in recent yearsPlasmodium species prevalent in a rural population in the area of Fougamou, Gabon. P. malariae and P. ovale infections  are commonly underreported as parasitemia is often low and severe disease is uncommon3. However, infections by these species can be chronic and contribute significantly to morbidity by e.g. causing anemia4. A previous study in the same area on symptomatic patients with malaria caused by non-falciparum/mixed species infections revealed an unexpected complexity of infections when analysis was done by deep sequencing technology5. Within the current study, the submicroscopic and microscopic prevalence of the different Plasmodium species in the resident population was investigated before we analysed in more depth the most virulent and abundant parasite P. falciparum by evaluating prevalence of gametocytes and the presence of chloroquine resistant genotypes.We did a cross-sectional survey to investigate and further characterize the different 6. Control and elimination strategies of malaria therefore need to employ highly sensitive techniques to identify such reservoirs. One approach is to assess P. falciparum gametocyte carriage by quantitative real-time PCR (qPCR) based on the marker Pfs25. In addition, molecular markers for drug-resistance and other polymorphisms can be analyzed to inform the best intervention strategies. E.g., in many African countries chloroquine sensitivity of P. falciparum returned after change of treatment policy and subsequent withdrawal of chloroquine from the market7, but this was not yet observed in Gabon9 despite discontinuation of chloroquine for malaria treatment in Gabon since 200310.Recently recognized as an important malaria transmission reservoir, asymptomatic infections and submicroscopic gametocytemia are major hurdles for malaria eliminationPlasmodium population infecting humans in Fougamou, Gabon. This is done by assessing prevalence of different Plasmodium species, P. falciparum gametocyte carriage and prevalence of P. falciparum chloroquine resistance by qPCR in a cross-sectional survey.With this study, we aimed at characterizing the whole Plasmodium parasites by Giemsa stained thick blood smear, 72 (8.6%) of them were considered as being mixed or non-falciparum infections. Microscopic results have been published before11. Body temperature was recorded for 771 individuals, mean temperature was 37.1 °C; 24 individuals presented with a body temperature ≥ 38 °C, 14 of those 24 had a parasitemia that could be detected by microscopy.We included 840 individuals and obtained blood of 834 persons, description of the study population is given in Fig. P. falciparum versus non-falciparum species was performed.PCR and microscopy reading results were well correlated . All PCR samples with a Cq < 17.7 (n = 85) were microscopically positive, and all samples with a Cq > 29 were microscopically negative (n = 76), hence below the limit of detection for microscopy. 214/217 (98.6%) PCR negative samples were also read negative by microscopy, three were read false positive with a low parasitemia (below 25 parasites/µl) confirming the accuracy of our microscopy results. Non-falciparum species were not further distinguished by microscopy, but reported as such, so that only differentiation of P. falciparum, 193 (23%) for P. malariae, 74 (8.9%) for P. ovale curtisi and 38 (4.6%) for P. ovale wallikeri. Parasitemia of individuals decreased with age  individuals were positive for 18 S plasmodial DNA, of these 553 (66.3%) were positive for rum, 193 3% for P.falciparum infections presented as mixed infections with P. falciparum. In five individuals, all four prevalent species were found at the same time. The different combinations of infections can be seen in Table P. malariae was the most abundant non-falciparum parasite, 90.6% (175/193) infections presented as coinfections with another species. We did not find any P. vivax infection in our samples.Most non-Plasmodium species that was non-falciparum. Median age of the P. malariae monoinfected individuals (n = 18) was 60 years (interquartile range 57.25–69.75 years) and therefore significantly older than for the individuals having a mixed infection of P. malariae with P. falciparum . For the other non-falciparum infections sample size was small, for P. ovale curtisi monoinfected individuals (n = 7) the median age was 42 years (IQR: 10–60 years) versus 14 years (IQR: 9–46 years) in individuals having a mixed infection of P. ovale curtisi with P. falciparum (n = 43). Only one individual aged 8 years had a P. ovale wallikeri monoinfection. In general, a trend towards higher age in mono-infected subjects is present.There were only 26/834 individuals who had a monoinfection of a Plasmodium positive samples, one can see that in all age groups an evenly high proportion of individuals was infected (around 70%) (Table When dividing the population in age groups and looking at the pan-%) Table . Only th%) Table .Table 2IP. falciparum infection status can be seen. All age groups were similarly infected, with a peak in the adolescents and less infections in the very young.When having a closer look at the non-falciparum infections, a similar pattern to the 109 of the subset of 223 individuals were positive for Pfs25 by qPCR, of these 70 were also positive by microscopy. Age of Pfs25 positive individuals was significantly lower (median age = 13 years) than age of non-carriers (median age = 35 years), . Though not significant, the odds of carrying gametocytes in infected individuals was 1.5  times higher in microscopy positive than submicroscopic participants showing the influence of asexual infection on gametocyte carriage.P. malariae coinfection neither affected gametocyte carriage rate for P. falciparum , nor gametocytemia .pfcrt haplotypes, 223 (66.4%) had CVIET haplotype only, 38 (11.3%) had CVMNK only, and 1 (0.3%) harbored a SVMNK monoinfection. The rest carried double and triple infections of wild and mutant haplotypes as; CVMNK and CVIET in 71 (21.1%), CVMNK and SVMNK in 2 (0.6%), and 1 triple infection (0.3%) as shown in Fig. Of 336 samples screened for the three Plasmodium species. Infection was evenly distributed over all age groups, with the exception that very young children were less infected maybe be due to increased care by the parents. Highest prevalence was found in children/adolescents between 5–20 years as typical for endemic areas13. There is no indication that immunity against malaria wanes in very old age in our study population. Results are in line with other studies from rural areas in Gabon showing no decline in malaria transmission in recent years14 but in contrast with findings from urban areas16 and results from the RTSS vaccine trial17 where a much lower prevalence of Plasmodium infections was detected.Analysis by highly sensitive qPCR of blood samples of a rural population in Gabon revealed an unexpected breath of infection with 74% of individuals being infected with any P. malariae being the most prevalent. Frequent multi-species infections have been shown for certain communities harboring, low-density parasitemias of several species3. We could not find a single P. vivax infection in the investigated population neither as mono nor as co-infections. In fact, P. vivax has never been reported from Gabon. This is in line with the dogma that P. vivax is not present in Central/Western African populations as they are largely Duffy negative and P. vivax usually cannot infect Duffy negative individuals. However, our finding contradicts with recent findings reporting P. vivax presence in many regions across malaria-endemic Africa including countries bordering Gabon even though only at very low frequencies19. Reports show that P. vivax is regularly infecting Duffy-negative individuals in Madagascar20, and is present at low frequencies across most malaria endemic regions in Africa, infecting Duffy positive and Duffy negative individuals22. One hypothesis is that African wild living apes are infected with closely related P. vivax strains, that could in rare cases infect humans23. We could not see this in our samples, even though in national parks close to our sampling area wild apes are present. However, maybe the distance to this region is too big, or our sample size was too small. Further studies on P. vivax in Africa as well as in Gabon are needed to further elucidate this question. P. malariae is the second most abundant human malaria parasite after P. falciparum in many African countries25. Generally, quartan malaria caused by P. malariae is regarded as benign malaria even though it has been associated with nephrotic syndromes especially in young children26 and more long-lasting and chronic illness associated with anaemia3. A recent hospital-based survey studied systematically clinical symptoms due to P. malariae in Papua New Guinea discovering that even though a rare disease, children hospitalized with P. malariae also developed severe symptoms and even died4. The authors concluded that P. malariae is associated with anemia and a similar risk of mortality when hospitalized compared to children hospitalized with malaria caused by another Plasmodium  species.Parasitemia was higher in very young children and was decreasing with age as expected for a population in a highly endemic area. Prevalence of non-falciparum infections was higher than expected. Nearly 50% of all infections presented as coinfections with a non-falciparum species, with P. malariae and P. ovalespecies is not well investigated. Hypnozoites of P. ovale. species infections in this area are usually not treated with primaquine/tafenoquine as glucose 6 phosphate dehydrogenase (G6PD) deficiency is prevalent in this populations, testing of G6PD-deficiency is currently not reliably possible in field settings and treatment without prior testing is prohibited28. A study in Gabon investigating the efficacy of artemether-lumefantrine for the treatment of non-falciparum and mixed species infections found good efficacy for this standard drug combination in this indication28. However, others found that the standard treatment for P. falciparum might not be ideal to clear non-falciparum parasites completely31.The burden of disease caused by P. malariae infected individuals indicated that immunity against P. malariae is also – as for P. falciparum – build up with age, as parasite load was higher in younger individuals than in older ones. However, these data have to be interpreted with a caveat because preamplification was done before the species-specific PCR. Monoinfections with non-falciparum species only rarely appeared and most P. malariae infections presented as coinfections with P. falciparum. Monoinfections of P. malariae (18/193) were mainly found in older individuals. We have no explanation for this finding but one hypothesis could be that even though immunity to P. malariae is built up with age similarly to P. falciparum, but this immunity with old age is less complete for P. malariae than for P. falciparum as there is less exposure due to lower parasite load/infection intensity/immune stimulation. Further studies are needed to elucidate this further.Correlation of qPCR parasitemia data with age of P. falciparum infections are commonly present and reported from different transmission settings34 showing some level of dependency with age34 seasonality35 and geographic area34. These asymptomatic infections are mostly submicroscopic (detected by molecular methods) and in some cases carry the transmission stages, gametocytes37. Half of the analyzed samples in our study cohort were positive for P. falciparum gametocytes showing a potential of high transmission intensity in the area. Similar to our finding, P. falciparum PCR-positive individuals had a 45% and 44% gametocyte carriage rate in a study conducted in Malawi35 and Kenya36, respectively. In several studies, gametocyte carriage was inversely associated with age of the infected person38; revealing that under-five year olds39 and school-age children35 were carrying more gametocytes than adults. We also observed that gametocyte carriage is more frequent in young children than in adults. Gametocyte density was similar in children and adults in our cohorts, though Coalson et. al reported a trend of lower gametocyte density in school-age children than other age groups35.In malaria endemic regions, asymptomatic P. falciparum parasite population. Similar results were found in previous studies showing that the dominant haplotype remains the resistant haplotype CVIET41. This is unexpected as CQ is no longer commonly available in the market for malaria treatment in the study area. However, the closely related drug (amodiaquine) that is given as P. falciparum first line treatment in combination with artesunate might play a role in selection of chloroquine resistant strains. While pfcrt CVIET is the most prevalent genotype in Africa, another resistance-associated genotype pfcrt SVMNT is rarely reported from Africa but more commonly found in South-America and some Asian countries42. The presence of SVMNT genotype was first reported in Tanzania in 2004 and later in Angola44. To our knowledge, this is the first time the presence of SVMNK genotype is detected in Gabon. SVMNT has been associated with the emergence of amodiaquine resistance45 and this finding highlights the need for continuous monitoring and re-evaluation of current therapy where amodiaquine, as in Gabon, is used in combination with artemisinin.The chloroquine resistant genotype remains high in this P. falciparum resistant alleles is consistently reported after the withdrawal of the treatment. In general, except in the case of P. vivax infection, CQ-resistance is rarely reported in non-falciparum infection. Delayed parasite clearance and treatment failures of non-falciparum species are usually attributed to the special biology of these parasites , also considering stage specific activities of the drugs46. One study from Indonesia showed CQ resistance in P. malariae infections47, but this was not more repeatedly reported. As chloroquine resistance was never widely reported from other areas in non-falciparum species (except P. vivax), we would speculate that chloroquine remains efficacious against non-falciparum parasites in this area.We did not evaluate the presence of CQ resistance in non-falciparum species in our study area where high level of Plasmodium infection in this rural population in Gabon. For the final aim of elimination and eradication, also submicroscopic infections as well as non-falciparum species have to be considered to be able to sustain malaria control efforts. Otherwise, rural areas will always convey a reservoir for future infections.Analysis by qPCR reveals the complexity of Ethics approval was obtained from the responsible ethics committee Comité d´Ethique Institutionel of the Centre de Recherches Médicales de Lambaréné. Signed informed consent was obtained from adults ≥ 18 years or the legal guardian in case of minors, assent was additionally obtained from adolescents ≥ 12 years. All methods were performed in accordance with relevant guidelines and regulations.Plasmodium infections in 840 individuals aged from 1–96 years in a rural area of Gabon (Fougamou and villages in the surroundings). The area is characterized by close proximity to primary rain forest and small-scale farming. We invited everybody who lived in the chosen area and was older than 1 year to take part in the study. Blood was collected by venipuncture in an EDTA blood tube (Sarstedt), and temperature was taken by axillary measurement. A Giemsa-stained thin and a thick blood smear was performed and analyzed by Lambaréné method reading 100 microscopic fields on the same day by microscopy. All malaria cases  that were diagnosed positive by thick blood smear were treated according to the national guidelines . Whole blood samples (500 µL) were collected mixed with 1,300 µL RNALater (Thermo Fisher Scientific) and stored at −20 °C until nucleic acid extraction was performed.We performed a cross-sectional study in February/March 2016 to assess the prevalence of later stabilized blood specimen were used for two types of extractions: either total nucleic acids (DNA and RNA) or total RNA automated in the QIAsymphony® SP system (Qiagen). Before total nucleic acids extraction with QIAsymphony DSP DNA kit, RNAlater solution was removed by centrifugation and packed erythrocytes were resuspended with 1X PBS to a final volume of 420 μL and extracted by the QIAsymphony® SP as per the manufacturer’s instructions.RNAP. falciparum gametocyte detection by RT-qPCR was done as reported earlier48 with some modifications. Briefly, frozen samples were thawed and half the volume of the blood-RNALater mix (900 µL) was separated, transferred to a 2-mL sample tube  and centrifuged for 3 min at 16,000 g. The supernatant was discarded and 300 µL buffer RTL Plus (Qiagen) supplemented with 1% (v/v) β-mercaptoethanol was added to the pellet and vortexed for 5 min before loading onto the QIAsymphony SP (Qiagen). RNA purification was automated using the QIAsymphony RNA Kit (Qiagen) according to the RNA CT 400 protocol provided with the instrument.Total RNA extraction for All data was entered and reviewed using Excel, further analyses were done with GraphPad Prism version 5 and R v3.5.0. R packages tidyverse and funModeling were used for data processing and generation of graphics. Correlation of data was analyzed by Spearman test, data were compared by Mann Whitney U test, lines were fitted by linear regression. A p-value smaller than 0.05 was considered statistically significant.Plasmodium infections was performed by pan-Plasmodium reverse transcription quantitative PCR (RT-qPCR) assay as described earlier28, with a lower limit of detection of 6 parasites/mL. The small subunit ribosomal RNA gene (18 S rDNA) as well as the expressed transcripts were co-amplified in a single step for high sensitivity detection of Plasmodium parasites. The amplicon was selected from a gene domain that is 100% conserved across human malaria species.Screening for A result was interpreted as positive if the quantification threshold cycle of the amplification curve was below 40 (cutoff Cq < 40). The assay setup was performed in a sterile PCR workstation and the pipetting robot  was used for sample and mastermix dispensing into the wells of a 384 plate. The qPCR assay was carried out in the LightCycler 480 II (Roche Applied Science).Plasmodium assay positives were first subjected to conventional PCR amplification using primers by Snounou49 before further utilized as template for the qPCR. A limited cycle PCR was performed in a 50 µL reaction volume containing 7.5 µL of nucleic acids extract, 300 µM of each primer (PLU5 and PLU6), 1X Qiagen PCR buffer with 1.5 mM MgCl2, 250 µM dNTPs each, and 1U Taq DNA Polymerase (Qiagen). The cycling conditions were: Initial denaturation at 95 °C for 5 min, followed by 20 cycles of  and a final extension at 72 °C for 5 min.Pan-P. falciparum, P. malariae, P. ovale curtisi, P. ovale wallikeri, and P. vivax) using primers and probes described previously28. Briefly, each of the species-specific qPCR assays consisted of 2.5 µl of the pre-amplified product, 1X SensiMix II Probe No-ROX (Bioline), 300 µM of each primer pair and 150 µM of each probe (Supplementary Table Plasmodium PCR positive but species PCR negative samples were repeated by a standard PCR using primers specific for cytochrome B50 (n = 16) or 18 S rRNA genes5 (n = 6) and subsequent Sanger’s sequencing for samples with a high enough parasitemia (Cq below 36). All but one  of the sequencing results revealed a P. falciparum infection.Single-plex qPCR assay was performed for five human malaria species  of samples was selected and analyzed by RT-qPCR for the presence of gametocytes in the infected participants. Gametocyte specific qPCR was performed to measure the transcript levels of Pfs25 mRNA using previously published targets, with modifications. A Taqman RNA-to-CT™ 1-Step kit (Thermo Fisher Scientific) was used for the quantitation of the gene expression levels. The reaction contains 1 × Taqman enzyme mix, 150 nM probe, 1 × Taqman RTqPCR mix, 400 nM of each primer and 2.5 µL of RNA extract.Among samples positive for 48 and are given in S1. Gametocyte carriage was only evaluated for P. falciparum and not for the other Plasmodium species.The reaction conditions consisted of reverse transcription (48 °C/20 min), enzyme activation (96 °C/10 min), and two-temperature cycling steps . The Cq value was determined as above for the species qPCR. All samples were tested in triplicates together with the non-RT and non-template controls. Samples were considered positive when a Cq value below 40 was present in at least two technical replicates. The used primer and probe sequences were published previouslyP. falciparum gametocytes. 300 microscopic fields were read using the Lambaréné method by two independent experienced microscopists.The samples positive by qPCR for Pfs25 were re-read by microscopy to check for 52. To enhance sensitivity, a preamplification followed by qPCR was done with primers listed in S1. In brief, a conventional PCR was carried out using PfCRT_Preamp1 & PfCRT_Preamp2 for 20 cycles in 25 μL reaction volume. Limited PCR-cycled products were used as templates in a qPCR multiplex reaction containing three hydrolysis probes. Each probe representing one of the three Pfcrt haplotypes; the CQ sensitive haplotype (CVMNK), and the two CQ resistance-associated haplotypes . Different fluorophores were tagged to each probe enabling detection of the haplotypes in parallel from each sample. DNA extracted from P. falciparum strains 3D7, Dd2, and 7G8 were used as positive controls for haplotypes CVMNK, CVIET and SVMNT, respectively.A multiplex qPCR assay was used to genotype Pfcrt gene spanning codons 72–76 using previously described hydrolysis probes and primers with modificationsSupplement


Border malaria, a shared phenomenon in the Greater Mekong Sub-region of Southeast Asia, is a major obstacle for regional malaria elimination. Along the China–Myanmar border, an additional problem arose as a result of the settlement of internally displaced people (IDP) in the border region. Since asymptomatic malaria significantly impacts transmission dynamics, assessment of the prevalence, dynamics and risk factors of asymptomatic malaria infections is necessary.Cross-sectional surveys were carried out in 3 seasons  and 2 sites (villages and IDP camps) in 2015. A total of 1680 finger-prick blood samples were collected and used for parasite detection by microscopy and nested RT-PCR (nRT-PCR). Logistic regression models were used to explore the risk factors associated with asymptomatic malaria at individual and household levels.Plasmodium infections was 23.3% by nRT-PCR, significantly higher than that detected by microscopy (1.5%). The proportions of Plasmodium vivax, Plasmodium falciparum and mixed-species infections were 89.6, 8.1 and 2.3%, respectively. Asymptomatic infections showed obvious seasonality with higher prevalence in the rainy season. Logistic regression analysis identified males and school children (≤ 15 years) as the high-risk populations. Vector-based interventions, including bed net and indoor residual spray, were found to have significant impacts on asymptomatic Plasmodium infections, with non-users of these measures carrying much higher risks of infection. In addition, individuals living in poorly constructed households or farther away from clinics were more prone to asymptomatic infections.The prevalence of asymptomatic Plasmodium infections were highly prevalent in the border human populations from IDP camps and surrounding villages. Both individual- and household-level risk factors were identified, which provides useful information for identifying the high-priority populations to implement targeted malaria control.Sub-microscopic  Anopheles mosquitoes [In the past decades, intensive malaria interventions have resulted in a dramatic decline in global malaria morbidity and mortality. However, the global burden of malaria is still enormous: in 2016 there were about 216 million cases resulting in ~ 445,000 deaths , 4. Compsquitoes –15. Withsquitoes . Since tsquitoes , 20, idePlasmodium reservoir has been reported to be considerable, even in low-transmission settings along the international borders in GMS, including Thailand–Cambodia, Thailand–Myanmar and China–Myanmar borders [Plasmodium vivax outbreaks in recent years [In the Greater Mekong Sub-region (GMS) of Southeast Asia, malaria distribution is heterogeneous and often concentrated along international borders –24. Bord borders , 27, 28. borders –31. In tnt years . The potPlasmodium infections as important reservoirs. The use of highly sensitive molecular detection methods allowed the detection of almost 20% of local residents as carriers of asymptomatic Plasmodium infections [With continued transmission of malaria in the China–Myanmar border, this study wanted to address the potential of asymptomatic fections . In the The study protocol was reviewed and approved by institutional review boards of China Medical University, China, Pennsylvania State University, USA and the local Health Bureau of Kachin State, Myanmar. Before carrying out the study, all adult participants or legal guardians of children voluntarily signed the informed consent.Anopheles minimus s.l. is the predominant vector species complex, and adult Anopheles density on average is higher in villages than IDP camps [This study was conducted in 4 villages  and 2 IDP camps (N2 and N3) near Laiza township (97.56°E and 24.75°N), Kachin State, Myanmar, along the China–Myanmar border area (π)(1 − π)/δ2, using an expected asymptomatic malaria prevalence for the border (π) of 20% [Sample size required to detect the prevalence of asymptomatic infection in the survey sites was calculated as n = (μ) of 20% ; μ stati) of 20% . During Thick and thin blood films were screened for the presence of malaria parasites using light microscopes with 100× oil immersion lens. Two microscopists examined all slides, and when discrepancy occurred, a third microscopist read the slides and the results were combined. For RNA extraction, 1 mL of Trizol reagent was added to the frozen sample containing 0.1 mL of blood. Total RNA was isolated according to the manufacturer’s protocol and dissolved in 30 μL RNase-free water. One microgram of each RNA sample was used to synthesize cDNA using the Takara RNA PCR kit (AMV) version 3.0 . Parasite detection was performed by nested RT-PCR (nRT-PCR) targeting the 18S rRNA using an established protocol [P < 0.25 were selected to be involved in the following multivariate analysis [P < 0.05 in multivariate analysis were considered to be the risk factors. Individual-level, independent variables included gender, age, occupation, malaria history, and bed net usage. Household-level factors included education of household head, building material of the house, IRS usage and location elevation. In addition, spatial location relative to landmarks was acquired for each household and considered household-level variables for analysis. For descriptive analyses, χ2 test or Fisher’s exact test was used. Asymptomatic malaria prevalence for the predictive factors was compared between the rainy and dry seasons, and between villages and IDP camps using χ2 test, and odds ratio (OR) and adjusted odds ratio (AOR) were calculated. P < 0.05 was considered statistically significant.Maps were generated using Google Earth. Statistical analysis was performed using SPSS Statistics 19.0. For risk factor analysis, both univariate and multivariate mixed effects logistic regression models were applied. First, explanatory variables were tested individually in univariate analysis. Then, all factors with analysis , 36. PotA census prior to the surveys identified the population size in the 4 study villages as 1436. The IDP camps were established in 2011 as the result of Myanmar civil war with an initial population of ~ 7000, which was increased to 10,402 in 2015 with populations migrated from more interior areas of Kachin. Using the prevalence of ~ 20% of asymptomatic infections determined using a sensitive molecular method , the samP. vivax infections in 25 samples, giving an overall 1.5% parasite prevalence. The prevalence of P. vivax infections in the peak season (2.3%) was slightly higher than that of the early season (1.7%) and late season (1.5%), but the differences were not statistically significant  Plasmodium falciparum, and 9 (2.3%) mixed P. vivax/P. falciparum infections. Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi infections were not detected. It is noteworthy that P. falciparum infections were only identified in the 2 later surveys.Microscopic examination of the 1680 blood smears only detected season 2.% was sliP = 0.001): 18.4% of infection occurred in early season (March–April), 30.1% in the rainy season (July), and 21.2% in the late season (November) (Table P < 0.001), whereas this trend was obviously reversed in the peak transmission season . This trend was maintained after the rainy season, and the prevalence of asymptomatic infections in the villages (26.3%) was much higher than that in the IDP camps (19.3%) in November, though the difference was marginal (P = 0.069).In contrast to the results from microscopic examination, there were significantly seasonal differences in the prevalence of infections detected by nRT-PCR  were selected and set into the final multivariate model. First, the results showed that males were significantly more likely than females to have asymptomatic infections . Second, the age group of ≤ 15 had significantly higher odds of malaria infection . Third, the effects of vector control-based preventive measures were very remarkable. Those who did not sleep under the bed nets had more than 5 times higher odds of asymptomatic infections than those who did . People from the households that did not have IRS had 3.4 times higher odds of asymptomatic infection than those with IRS . Fourth, major building materials of the houses, which reflect the economic status of the family, also showed significant effect on asymptomatic infections. Residents from the houses constructed from wood or bamboo had approximately twice the odds of having Plasmodium infections . Last, longer distance of household to the nearest clinic (> 180 m) was also significantly associated with a higher risk of malaria infection   and 3.81 times  higher odds of having asymptomatic infections than those who used bed nets in the rainy and dry season, respectively (Table P < 0.001). Similarly, those without the use of IRS had 7.78  and 2.29  times higher odds of infection in the rainy and dry season, respectively (Table P < 0.001). Furthermore, larger distance (> 180 m) of the household to the nearest clinic was significantly associated with a higher risk of malaria infections in both seasons. For other factors, there were also seasonal variations. Whereas the male gender had increased odds of asymptomatic infections in the rainy season, the factor did not seem to contribute to higher infection prevalence in the dry season  were combined and compared with that conducted in the rainy season (July). With regard to preventive measures, those who did not use bed nets had 10.53  than the non-users from the villages . Similarly, IRS use also significantly reduced the odds of asymptomatic infections in households from both villages and camps, but the effect appeared more profound in the villages  were significantly associated with asymptomatic infections. Such a gender bias of malaria infection has been reported in other studies , 46–49. lciparum , which mMalaria transmission is mainly influenced by two key factors: gametocyte infectivity and vector density. Thus, effective vector-based malaria control measures such as the use of IRS and bed nets, especially long-lasting insecticide-treated nets (LLINs) are particularly important in reducing malaria incidence . This stThis study also found that household and the surrounding environmental factors affected the prevalence of asymptomatic malaria. Firstly, houses constructed with wood or bamboo increased the risks of infection, corroborating findings from other studies –62. SincThe findings of these 3 seasonal cross-sectional surveys have provided insights about persistent malaria transmission along the China–Myanmar border, which is needed for malaria elimination on both sides of the border. A sensitive molecular method verified the presence of a large submicroscopic reservoir of malaria parasites in both village and IDP populations. This study also identified risk factors associated with increased prevalence of asymptomatic infections in both populations. On the individual level, males, school children, and those who did not use bed net were the as high-risk populations. On the household level, worse living conditions, failure to use the mosquito control-based interventions and farther away from the clinic were also associated with increased risks of parasite infections. While increasing the coverage of effective malaria interventions will be the key to control border malaria, additional measures such as mass drug administration (MDA) may be considered in order to reduce the infected population. MDA with chloroquine and primaquine has been found to be successful in eliminating vivax malaria in central China . FurtherThis study has several limitations. First, the data collection was confined to the available residents in the households, which led to the female-biased study population and over-representation (54.6%) of office/in-house workers, which undermines the generalization of the findings. Given that males, farmers and soldiers might be the high-risk populations, future studies should ensure a better representation of the study population. Second, the use of convenience samples inevitably adds additional biases, and future study designs need to be more systematic. Third, the study did not take into account the effect of human migration on malaria transmission. It is important to find out whether and how much migration of people from more malaria-endemic interior areas to the camps contributes to the malaria transmission. Furthermore, the significance of cross-border migration for malaria importation to the neighbouring Yunnan province of China also deserves special attention. Finally, the comparison between the local villages and camps needs to be more quantitative since their accesses to malaria treatment and prevention services were different. Because the whole region is targeted for malaria elimination, provision of quality malaria control services to the entire populations will ensure achieving the goal.This study underscores the need for strong initiatives to control border asymptomatic malaria. Results from this study provide the needed knowledge to the malaria managers for defining the priority populations and areas for more intensified control efforts. Particularly, vector-based control measures such as bed nets and IRS appeared to provide significant protection against asymptomatic infections, and thus attention is needed to increase the coverage of these measures.


Subpatent malaria infections, or low-density infections below the detection threshold of microscopy or standard rapid diagnostic testing (RDT), can perpetuate persistent transmission and, therefore, may be a barrier for countries like Namibia that are pursuing malaria elimination. This potential burden in Namibia has not been well characterized.Using a two-stage cluster sampling, cross-sectional design, subjects of all age were enrolled during the end of the 2015 malaria transmission season in Zambezi region, located in northeast Namibia. Malaria RDTs were performed with subsequent gold standard testing by loop-mediated isothermal amplification (LAMP) using dried blood spots. Infection prevalence was measured and the diagnostic accuracy of RDT calculated. Relationships between recent fever, demographics, epidemiological factors, and infection were assessed.Plasmodium falciparum malaria infection was low: 0.8% (16/1919) by RDT and 2.2% (43/1919) by LAMP. All but one LAMP-positive infection was RDT-negative. Using LAMP as gold standard, the sensitivity and specificity of RDT were 2.3% and 99.2%, respectively. Compared to LAMP-negative infections, a higher portion LAMP-positive infections were associated with fever , though 55% of infections were not associated with fever. Agricultural occupations and cattle herding were significantly associated with LAMP-detectable infection , while gender, travel, bed net use, and indoor residual spray coverage were not.Prevalence of This study presents results from the first large-scale malaria cross-sectional survey from Namibia using molecular testing to characterize subpatent infections. Findings suggest that fever history and standard RDTs are not useful to address this burden. Achievement of malaria elimination may require active case detection using more sensitive point-of-care diagnostics or presumptive treatment and targeted to high-risk groups.The online version of this article (10.1186/s12936-018-2626-5) contains supplementary material, which is available to authorized users. Subpatent malaria infections, or low-density malaria infections below the detection threshold of standard rapid diagnostic tests (RDT) or microscopy, may be responsible for at least 20–50% of malaria transmission in pre-elimination and elimination settings –4. As suA better understanding of the prevalence and characteristics of subpatent infections can inform the design of targeted strategies for malaria elimination –9. NamibPlasmodium falciparum and the most common malaria vector species is Anopheles arabiensis [Zambezi region is located in the northern malarious area of Namibia, and shares borders with the higher malaria endemic countries of Angola and Zambia Fig. a. It is abiensis .Fig. 1a This cross-sectional survey was carried out at the end of the transmission season from April to June 2015 in the historically higher burden western part of Zambezi region , annual parasite incidence (API) of at least 15/1000, 40% probability of clinical symptoms, 51% probability of seeing care at a health facility if febrile, 85% RDT diagnostic sensitivity for symptomatic infections, and 50% prevalence of annual incidence infections during the survey , 17. AssAll individuals in selected households were eligible for inclusion in the survey if they were usual residents of the household or had slept in the household the night before. A complete line listing of all eligible individuals was generated at the first visit and subjects were excluded if they refused to participate, were too ill to participate, or were not present at any of three visits to each household.™ Malaria HRP2/pLDH (Pf/PAN) RDT  and to generate a dried blood spot (DBS) using Whatmann™ 3 MM filter paper . Presence of clinical symptoms was assessed by self-report of fever in the prior 2 weeks. Infections associated with lack of self-reported fever were considered subclinical. Participants were interviewed in the local language SiLozi using a standardized tablet-based household questionnaire (administered to the household head or other adult representative) and individual questionnaire via Open Data Kit (ODK) software.Selected households were approached for written informed consent, which was conducted at the individual level and from a parent or guardian for minors less than 18 years of age. Additionally, minor assent was obtained for children > 12 years of age. Blood was collected by finger prick from all consenting participants for malaria testing by CarestartHousehold level data included the household location, socioeconomic status, household population, ITN ownership, as well as IRS coverage in the past 12 months. Questions assessing socioeconomic status included assessment of water source and household assets including electricity, radios, televisions, mobile phones, and refrigerators. The individual questionnaire assessed demographics, travel history, vector control coverage and use, history of malaria, and care-seeking behaviour associated with fever the past 2 weeks. All RDT positive individuals were considered to have uncomplicated malaria and received treatment in the field with artemether-lumefantrine according to national guidelines . SubjectPlasmodium-specific testing was conducted using a commercial loop-mediated isothermal amplification kit  in accordance with manufacturer’s instructions [AluI digest for nPCR positives [DBS cards were transported to the University of Namibia Multi-Disciplinary Research Center laboratory in Windhoek. DNA was extracted using the Saponin/Chelex method previously described . Using 1ructions . For quaositives . Five miSurvey data were uploaded from tablet devices to a secure cloud-based sever at least weekly. Data were analysed using STATA . Locations of cases were mapped using QGIS (version 2.16.2). Prevalence of malaria infection by RDT and LAMP were estimated and compared. Diagnostic accuracy of RDT was calculated using LAMP as gold standard. Based on the nPCR results of 10% of negative cases and all discordant LAMP and RDT samples, quality assurance of LAMP was assessed by calculating sensitivity and specificity of LAMP using nPCR as a gold standard. Demographic and epidemiological characteristics of participants were described. A housing quality variable was constructed by a principal component analysis based on type of wall, roof, window, and eaves. A socioeconomic status index was generated by principal component analysis based on six household assets ownership variables, wall type, roof type, and household water source.p value of less than 0.10 in this bivariate analysis were included in multivariate logistic regression models, with gender, age, and socioeconomic status included a priori. Comparisons accounted for clustering at the highest level [Associations between covariates and LAMP-detectable malaria infection were explored using Chi squared and Fisher’s exact tests Table . Epidemist level , 23  were enrolled . By LAMP, prevalence was 2.2%, 95% CI 1.6% to 2.9% (43/1919) and in subsequent nPCR testing, all LAMP-positive infections were identified as P. falciparum mono-infection. Malaria prevalence by health facility catchment area ranged from 0 and 1.7% by RDT and 1.5 to 2.8% by LAMP. There were no statistically significant differences in malaria prevalence by health facility catchment area .The overall prevalence of Figure Compared to 2.2% infection prevalence by LAMP, prevalence of infection by RDT was only 0.8%. There was little concordance between RDT and LAMP positivity, with only one participant positive by both detection methods. Of seven symptomatic individuals who were RDT positive and LAMP negative, 2 (28%) reported treatment with artemether–lumefantrine within the past 2 weeks. Using LAMP as gold standard, the diagnostic accuracy of RDT was assessed in the total study population and in those with and without fever in the previous 2 weeks  and specificity was 99.2% (95% CI 98.6–99.5), respectively. Among the samples that had repeat nPCR results, there were no discordant nPCR results. There was only one LAMP negative sample that was PCR positive. That all but one of the 15 RDT-positive LAMP-negative samples were confirmed negative by nPCR suggests that these samples were false RDT-positives , only 51.8% (95% CI 48.4% to 55.3%) reported seeking care .


The aim of the proposed study was to find out discriminating clinical features of Falciparum malaria was diagnosed by looking at the ring and gametocyte stages by microscopic examination in Giemsa stained slides. Dengue was diagnosed by ELISA for dengue-specific IgM and IgG. Liver enzymes (AST and ALT) and kidney markers (creatinine and urea) were estimated by standard biochemical techniques.falciparum malaria. Acute renal failures and neurological sequelae were noticed in slightly higher number of dengue patients.AST and ALT showed similar rise in both, severe malaria and dengue patients but it was much pronounced in dengue haemorrhagic fever where it attained 3–4 folds increase. Creatinine and urea showed higher levels in dengue compared to malaria. Thrombocytopenia (76.27%), convulsions (18.64%) and hepatic dysfunction (5.08%) were more prominent in dengue than that in malaria where these parameters were 50.89, 7.14 and 2.67%, respectively. Conversely, cases with anaemia, splenomegaly and jaundice were three times more in falciparum malaria. Neurological sequelae and cases of acute renal failure were almost equal in both the infections.Thrombocytopenia and hepatic dysfunction were more common in dengue, while anaemia, splenomegaly, jaundice and convulsions were more frequent in  Aedes aegypti to around 50 million people of 2.5 billion population at risk throughout the world  is the causative agent of dengue and is generally transmitted by –4 is thehe world . PatientPlasmodium falciparum  antigen detection, ELISA was performed in patients who had fever for 3–5d, while those who had high fever for more than 5 d after the onset of illness were tested for dengue specific IgM and IgG. Serological studies were performed with the qualitative dengue IgM capture ELISA and dengue indirect IgG ELISA . Dengue patients were treated with acetaminophen and symptomatic therapy in the form of fluid and antipyretics whereas patients suffering from falciparum malaria were advised artesunate, as chloroquine and fansidar are not responding in a fairly good number of cases in our experimental areas.Patients with fever, headache, convulsions, body ache and haemorrhagic manifestations were tested for dengue fever (DF) and dengue haemorrhagic fever (DHF). Clinical symptoms such as high fever, headache, thrombocytopenia, splenomegaly, convulsions and neurological sequelae were observed and compared in the confirmed cases of Thrombocyte counts were recorded in both malaria and dengue patients. For the estimation of liver and kidney markers, blood was collected in sterile glass test tubes by vein puncture of the malaria and dengue patients after taking their consent. These samples were allowed to clot at room temperature and then transferred to refrigerator at 5 °C for 12h to squeeze the clot. Serum was collected and centrifuged at 2000rpm for 5min to remove probable contaminations and finally stored in sterilized vials at −80 °C until analyzed for liver enzymes and kidney markers. Aspartate transaminase (AST) and alanine transaminase (ALT) were estimated , while cFollow up of all the patients was done till they were discharged from the hospital. All the dengue and malaria cases were monitored for their clinical, biochemical and haematological profiles. Clinical parameters of dengue and malaria positive patients were studied and compared to observe prominent discriminating features of these infections.All clinical parameters were examined by chi-square test using GraphPad InStat 3.06 software , and the level of significance was set at P< 0.05. The results were expressed as mean ± standard deviation (SD) using SPSS . Graphs were plotted on Sigma plot 12.0.P. falciparum, while 120 (51.72%) of P. vivax. As for dengue is concerned, 183 suspected cases having high fever, headache and body ache were examined during its active transmission season in 2014–15 from the northeastern region of India and Makkah region of Saudi Arabia, out of which 59 (32.77%) were found positive for dengue. Majority of the patients 47 (80%) were of classical dengue, while 12 (20%) patients showed the symptoms of dengue haemorrhagic fever. Overall, 40 (67.79%) patients were found positive for IgM and 12 (20.33%) were positive for IgG. A total of 26 (44.06%) patients showed both IgM and IgG. During illness, gum bleeding and skin petechiae were seen in 3.39 and 6.80% patients, respectively.During peak season, 380 suspected cases with high fever and headache were examined for malaria from Aligarh district during 2014–15, out of which 232 (61%) were positive for malaria. Out of 232 positive cases 112 (48.27%) patients were of falciparum malaria and dengue patients , in which parasites reappeared after 23 days, indicating resistance as recrudescence occurred. Symptoms in patients infected with P. falciparum and dengue showed significant differences in skin rash, sweating, vomiting and body ache, but the values relating fever, headache, nausea, dizziness and abdominal pain were very close in both the diseases and did not show significant differences . Convulsions and hepatic dysfunction were more common in dengue (18.64 and 5.08%) than in malaria (7.17 and 2.67%). In contrast, jaundice was more common in malaria patients (6.25%), than in dengue (1.70%). Acute renal failures and neurological sequelae were also slightly more pronounced in falciparum malaria compared to dengue in terms of percentage but the difference was not significant  having low to moderate infections. Slightly raised values of AST  and ALT  were recorded in severe cases of falciparum malaria and dengue fever, respectively. Almost five times increase in the level of AST (215.50±41.71IU/L) and three times in ALT (176.00±19.79IU/L) were recorded in severe dengue haemorrhagic fever followed by mild cases of DHF where AST and ALT were recorded as 108.00±32.35 and 75.33±12.66IU/L, respectively. However, these enzymes remained within normal range in light infections.Liver damage was observed as a common complication in both malaria as well as dengue infections. Liver function test (LFT) was performed by estimating serum aspartate transaminase (AST) and alanine transaminase (ALT). The normal values of these enzymes in applied technique were 0–45IU/L and 3–60IU/L for AST and ALT, respectively. Both liver enzymes and kidney markers were almost within normal range in P. falciparum and dengue infections were assessed by measuring elevated creatinine and urea in serum. Patients suffering from falciparum malaria with high parasitemia, severe dengue and dengue haemorrhagic fever showed an increase in the level of creatinine to 158.76±6.99, 186±11.50 and 187.00±5.65μmoles/L, respectively compared to the normal range of 72–126 μmoles/L, while it was within range in low and mild infections with the exception of DHF, which showed a slightly raised value (135.33±25.38μmoles/L) in mild DHF patients. Serum urea too showed slightly higher values in severe cases of malaria, DF and DHF, which were recorded as 9.60±1.32, 8.10±0.91 and 10.20±1.97mmoles/L against the normal range of 3.0–6.0mmoles/L . Headache and nausea in dengue recorded in 75 and 69% patients in Makkah region  in our study.All the malaria, dengue and dengue haemorrhagic fever patients had high fever as earlier observed for these infections in Jazan and Makkah region of Saudi Arabia, Pakistan and India –21. In oh region  were almh region  which wafalciparum malaria and was noted in 50.89% patients in our study which is almost same as earlier observed in Pakistan where 54.5% cases of P. falciparum had thrombocytopenia  in P. falciparum infection were recorded in Saudi Arabia  compared to dengue where it was observed only in 10.17 % cases. Higher rate of splenomegaly in falciparum malaria was probably due to phagocytosis of parasitized R.B.Cs which got accumulated in the spleen for clearance. Almost similar enlargement in around 39–45% P. falciparum malaria patients was observed in Saudi Arabia  was reported in Vietnam  were recorded for mild P. falciparum infections in Nigeria  in 7.14% P. falciparum. Comparatively, much higher rate of ARF of 19% was recorded from the patients in Pakistan  is a common cause of morbidity and mortality in severe Pakistan . Low ratPakistan , 43, 44.falciparum malaria. Immune mediated mechanism and direct tropic effect of virus are implicated in neurological manifestations, as dengue antigen has been demonstrated in the brain of few patients with dengue encephalitis  than in malaria (7.14%) indicating more neurological damage in dengue than in phalitis . We obsend India , 46–48.  in Mali . Like defalciparum malaria. Neurological sequelae and cases of acute renal failure were almost equal in both the types of infections.Thrombocytopenia and hepatic dysfunction were more common in dengue, whereas anaemia, splenomegaly, jaundice and convulsions were more frequent in


Malaria in pregnancy has major impacts on mother and child health. To complement existing interventions, such as intermittent preventive treatment and use of impregnated bed nets, we developed a malaria vaccine candidate with the aim of reducing sequestration of asexual “blood-stage” parasites in the placenta, the major virulence mechanism.Plasmodium falciparum protein responsible for binding to the placenta via chondroitin sulfate A (CSA). Healthy, adult malaria-naive volunteers were immunized with 3 intramuscular injections of 20 μg (n = 9) or 50 μg (n = 27) PAMVAC, adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) or in a liposomal formulation with QS21 (GLA-LSQ). Allocation was random and double blind. The vaccine was given every 4 weeks. Volunteers were observed for 6 months following last immunization.The vaccine candidate PAMVAC is based on a recombinant fragment of VAR2CSA, the P. falciparum-infected erythrocytes to CSA in a standardized functional assay.All PAMVAC formulations were safe and well tolerated. A total of 262 adverse events (AEs) occurred, 94 (10 grade 2 and 2 grade 3) at least possibly related to the vaccine. No serious AEs occurred. Distribution and severity of AEs were similar in all arms. PAMVAC was immunogenic in all participants. PAMVAC-specific antibody levels were highest with PAMVAC-GLA-SE. The antibodies inhibited binding of VAR2CSA expressing PAMVAC formulated with Alhydrogel or GLA-based adjuvants was safe, well tolerated, and induced functionally active antibodies. Next, PAMVAC will be assessed in women before first pregnancies in an endemic area.EudraCT 2015-001827-21; ClinicalTrials.gov NCT02647489. The pregnancy-associated malaria-vaccine candidate PAMVAC is safe and well tolerated with all three tested formulations and induces functional binding-inhibitory antibodies. The vaccine with glucopyranosyl lipid adjuvant (GLA) in stable emulsion is superior to Alhydrogel and a GLA/QS21 liposomal formulation. Plasmodium falciparum, semi-immunity develops over time, such that young children, especially, are at highest risk of developing severe malaria. During pregnancy, the previously acquired immunity is no longer sufficient, and significant morbidity and mortality of the mother and the child may occur. It is estimated that pregnancy-associated malaria (PAM) results in 20 000 maternal and 200 000 infant deaths annually [The vast majority of malaria episodes occur in sub-Saharan Africa. With continuous exposure to the main responsible parasite, annually .Plasmodium falciparum–infected red blood cells (iRBCs) sequester in the vasculature including the placenta [P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, which are expressed on the surface of iRBCs [var genes that code highly variable PfEMP1 proteins, mediating binding to multiple different endothelial receptors [P. falciparum occur [placenta  and therof iRBCs . Each paeceptors . Broadlyeceptors . One memeceptors . Placenteceptors . Thus, peceptors  when sigum occur , 12. Theum occur . The maium occur , 18. In um occur , 19–26.The preferred product characteristic of a placental malaria vaccine is induction of lifelong protection with 1 dose given to adolescent girls. This could be given together with the human papillomavirus vaccine to adolescent girls and would have several advantages over the World Health Organization’s recommended methods that consist of intermittent treatment, bed nets, and prompt diagnosis. Long-lasting vaccines offer a much greater window of protection than any chemoprophylactic intervention. Vaccine-induced protection can be initiated before the first pregnancy, supported by bed nets at a critical time period because pregnancy could proceed unnoticed or unreported . HoweverFollowing extensive evaluation, the lead candidate PAMVAC was identified . It spanHere, we report results of the first-in-human phase 1 clinical trial of 20 μg or 50 μg PAMVAC, formulated with Alhydrogel, GLA-SE, or GLA-LSQ in malaria-naive volunteers in Tübingen, Germany. Importantly, the antibodies acquired were functional. The activity was influenced by formulation in particular GLA-SE were highly immunogenic.The study was conducted in a malaria-naive cohort in Tübingen, Germany. The study was done concurrently with a trial in Cotonou, Benin, as part of a 2-center, randomized, staggered, adjuvant-selection, dose-escalation clinical phase 1 trial. Eligible volunteers were immunized with PAMVAC, adjuvanted with Alhydrogel, GLA-SE, or GLA-LSQ 3 times at 4-week intervals. Vaccinations were given by intramuscular injection in the deltoid. Injections were given on alternating sides, starting with the nondominant arm. Neither the clinical team nor the volunteers were aware of allocation to the Alhydrogel, GLA-SE, or GLA-LSQ arm. An independent pharmacist prepared the syringes with masking tape. Vaccinations started in 3 sentinel volunteers, receiving 20 μg PAMVAC adjuvanted with Alhydrogel, GLA-SE, or GLA-LSQ, respectively, followed by 2 volunteers in each group 1 day later (n = 3 per arm). At least 4 weeks later, the second group receiving 3 times 50 μg PAMVAC was started. The same procedures were applied as in the first group. All volunteers were examined by a physician 7 and 28 days following each vaccination. In addition, volunteers were contacted actively by the clinical team 1 and 14 days following each vaccination. Long-term follow-up visits were performed 16 and 28 weeks following the last vaccination (days 168 and 252). All volunteers were encouraged to contact the clinical team in case of any adverse event (AE). During the study period, the team was available 24 hours, 7 days a week.The primary endpoint of the trial was the number and grade of AEs and serious AEs (SAEs) possibly, likely, or definitely related to vaccination. Severity of AEs was graded as mild (grade 1), moderate (grade 2), severe (grade 3), or potentially life threatening (grade 4). For laboratory and solicited AEs, adapted Brighton Collaboration  and FoodThe trial was approved by the ethics committee of the Medical Faculty and the University Clinics of the University of Tübingen and the German Regulatory authorities. It strictly adhered to International Council for Technical Requirements for Human Use guidelines and the principles of the Declaration of Helsinki.Drosophila Schneider 2-derived cell line . Cells were expanded in shakers and Cultibags for 3 weeks. All medium components used for the upstream process were free of animal components. After 3 days in the production bioreactor, the bulk harvest was clarified by depth filtration, and the product was purified by a multistep process including SP Sepharose chromatography, virus removal (low pH treatment), Capto Adhere chromatography, Sartobind Q membrane polishing, Planova 20 N virus removal filtration, and an ultrafiltration/diafiltration step to reach the final concentration of 0.4 mg/mL. The drug product was aseptically vialed at Miltenyi (Germany) and stored at –20°C. The bulk batch of Alhydrogel was produced at Brenntag (Denmark), diluted, and filled at Nova Laboratories (United Kingdom) under cGMP and stored at 2°C–8°C. GLA-SE and GLA-LSQ were manufactured by the IDRI and stored at 2°C–8°C. The vaccine was prepared extemporaneously and passed to the clinical team in a syringe containing either 20 μg or 50 μg PAMVAC. The 20-μg PAMVAC preparations contained 0.17 mg Alhydrogel or 1 μg GLA with either 2% squalene (GLA-SE) or 0.4 µg QS21 (GLA-LSQ); the 50-μg PAMVAC contained 0.43 mg Alhydrogel or 2.5 μg GLA with either 2% squalene or 1 µg QS21, respectively.The PAMVAC vaccine candidate consists of an antigen encompassing the ID1-DBL2-ID2a subunits of the FCR3 variant of VAR2CSA . The vacPAMVAC-specific IgG was measured by indirect enzyme-linked immunosorbent assay (ELISA). Specific IgG present in the plasma bind to the ID1-ID2a coated onto the microplate wells and detected by horseradish peroxidase (HRP) conjugated goat anti-human IgG antibody. HRP activity was measured by color conversion of the substrate o-phenylenediamine dihydrochloride in the presence of hydrogen peroxide. Absorbance at 490 nm was measured after stopping the reaction with 0.5 M sulfuric acid. The optical density at different plasma dilutions was measured with a SpectraMax plate reader using SoftMax Pro GxP software. The level of PAMVAC-specific plasma IgG was defined as the area under the curve (AUC) of the sigmoid titration curve of 11 two-fold dilutions starting from 1:100. Titration curves were made for each volunteer at each day of study . The binding to heterologous ID1-ID2a variants was measured using a single dilution (1:400).P. falciparum ring-stage iRBC were exposed to tritium-labeled hypoxanthine for 24 hours, after that parasites were on late-stage trophozoite and schizont stages. A total of 200 000 cells per well were incubated for 2 hours at 37°C in a 96-well plate  coated the day before with 2 µg/mL CSA (D8428 Sigma-Aldrich) and blocked with 1% immunoglobulin-free bovine serum albumin (A7030 Sigma-Aldrich). Wells were run in triplicate either without plasma or with 4 two-fold dilutions from 1:10 of a pool of plasma from malaria-naive, a pool of plasma samples from malaria-exposed multiparous women from Benin [The ability of immunoglobulins to interfere with the binding of iRBC expressing native VAR2CSA was measured as previously described . BrieflyEligible participants were randomized on the day of first immunization by an independent party that used a computer-generated allocation table with participant identification codes. Sealed envelopes were provided to the pharmacist immediately before vaccination. The study was an exploratory phase 1 clinical trial. Hence, sample size was chosen to allow detection of large differences in AE pattern and PAMVAC-specific IgG concentration; that is, with 9 participants per group, 10% vs 80% of at least possibly related AEs and a difference in normally distributed variables larger than 60% of the standard deviation could be detected with 90% power and 5% alpha. Except for predefined immunological analyses, all statistical modeling was exploratory. A 2-tailed alpha <5% was considered statistically significant. All exploratory immunological analysis included dose and allocation as variables  if not otherwise stated. Clinical and demographic data were captured using OpenClinica, version 3.9.1; calculations were done using R, version 3.4.3.The study took place between 4 May 2016 and 9 March 2017. In total, 36 volunteers were recruited, 9 received 20 µg and 27 received 50 µg PAMVAC. Immunization was performed between 9 May 2016 and 30 August 2016. All vaccinations were given within the prespecified time window of 28–35 days. One volunteer, allocated to 50 μg PAMVAC-Alhydrogel, did not receive the third injection due to travel abroad. This volunteer remained in the study and follow-up continued until the last visit. One volunteer, allocated to PAMVAC-GLA-SE, missed the last follow-up visit (D252) because of relocation .2  and 1 with 18.3 kg/m2.Baseline demographics were similar between the groups , which rIn general, PAMVAC was well tolerated . No SAEsPAMVAC induced seroconversion in all volunteers irrespective of dose level and adjuvant . HoweverTime to peak PAMVAC-IgG concentration was shorter in volunteers vaccinated with PAMVAC-GLA-SE  and PAMVAC-GLA-LSQ  compared to PAMVAC-Alhydrogel. A comparison of peak concentrations instead of total PAMVAC-IgG at 4 weeks following last immunization (day 84) showed a similar fold difference in concentration of GLA-based compared to Alhydrogel-based adjuvants.P = .0005; The functional response that protects against placental malaria may include many effector mechanisms. However, an antibody response that hinders iRBC sequestering, thereby alleviating detrimental downstream inflammation events that take place in the placenta, appears the most plausible and well-documented mechanism of protection –33. Our Plasmodium reichenowi variant being least similar to the vaccine antigen; Due to substantial sequence variability even in the minimal binding domain of VAR2CSA, we investigated the level of cross-reactivity of PAMVAC-induced antibodies on day 84 by ELISA. We found that immunized volunteers acquired substantial levels of antibodies with specificity for the PAMVAC region of genetic variants representing different clades of VAR2CSA . The higP. falciparum malaria. The vaccine was well tolerated with only 2 transient and at least possibly related grade 3 AEs. One volunteer experienced an episode of fever on the day of first injection, and another developed an injection-site swelling. In contrast to preclinical findings in rodents and rabbits demonstrating induction of high levels of antibodies using GLA-LSQ compared to Alhydrogel or GLA-SE (see P. reichenowi genome. Again, the GLA-SE formulation showed the highest cross-reactivity.To our knowledge, this is the first report of a clinical trial testing a vaccine to generate an antibody response against the molecular determinants of a virulence factor of A-SE see , we founCurrently, there is no valid surrogate measure for protection against placental malaria . ValidatPrevious phase 1 studies with malaria vaccines have shown that immune responses can be significantly lower in endemic populations, but it is also conceivable that vaccine responses may be augmented by previous exposure , 37. TheIn conclusion, PAMVAC formulated with Alhydrogel, GLA-SE, or GLA-LSQ is well tolerated in healthy, malaria-naive adults. The antigen induces a functional IgG response and is most immunogenic when formulated with GLA-SE.Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.ciy1140_suppl_Supplementary_Figure_S1Click here for additional data file.ciy1140_suppl_Supplementary_Figure_S2Click here for additional data file.ciy1140_suppl_Supplementary_Figure_S3Click here for additional data file.ciy1140_suppl_Supplementary_Figure_S4Click here for additional data file.ciy1140_suppl_Supplementary_Table_1Click here for additional data file.ciy1140_suppl_Supplementary_Figure_LegendsClick here for additional data file.