Considerable success in reducing malaria incidence and mortality has been achieved in Brazil, leading to discussions over the possibility of moving towards elimination. However, more than reporting and counting clinical cases, elimination will require the use of efficient tools and strategies for measuring transmission dynamics and detecting the infectious reservoir as the primary indicators of interest for surveillance and evaluation. Because acquisition and maintenance of anti-malarial antibodies depend on parasite exposure, seroprevalence rates could be used as a reliable tool for assessing malaria endemicity and an adjunct measure for monitoring transmission in a rapid and cost-effective manner.This systematic review synthesizes the existing literature on seroprevalence of malaria in the Brazilian Amazon Basin. Different study designs  with reported serological results in well-defined Brazilian populations were considered. Medline (via PubMed), EMBASE and LILACS databases were screened and the articles were included per established selection criteria. Data extraction was performed by two authors and a modified critical appraisal tool was applied to assess the quality and completeness of cross-sectional studies regarding defined variables of interest.Plasmodium falciparum, 14 papers assessed Plasmodium vivax and four articles reported results on both Plasmodium species. Considerable heterogeneity among the evaluated malarial antigens, including sporozoite and blood stage antigens, was observed. The majority of recent studies analysed IgG responses against P. vivax antigens reflecting the species distribution pattern in Brazil over the last decades. Most of the published papers were cross-sectional surveys (73.9%) and only six cohort studies were included in this review. Three studies pointed to an association between antibodies against circumsporozoite protein of both P. falciparum and P. vivax and malaria exposure. Furthermore, five out 13 cross-sectional studies evidenced a positive association between IgG antibodies to the conserved 19-kDa C-terminal region of the merozoite surface protein 1 of P. vivax (PvMSP119) and malaria exposure.From 220 single records identified, 23 studies were included in this systematic review for the qualitative synthesis. Five studies reported serology results on P. falciparum and P. vivax exposure in areas with variable and unstable malaria transmission in Brazil. However, this study highlights the need for standardization of further studies to provide an ideal monitoring tool to evaluate trends in malaria transmission and the effectiveness of malaria intervention programmes in Brazil. Moreover, the score-based weighted tool developed and used in this study still requires further validation.This systematic review identifies potential biomarkers of  Plasmodium falciparum elimination targets, the species responsible for <15% of all malaria cases diagnosed in the Brazilian Amazon Basin.Recent emphasis and constant efforts on malaria elimination and eradication goals are changing the global malaria epidemiology scenario. Indeed, there has been an extraordinary scale-up of malaria control interventions over the last decade, which resulted in a substantial decline in global malaria morbidity and mortality . In thisBecause past and current malaria control policies and implementation strategies have successfully reduced transmission in Brazil, the measurement of malaria-associated morbidity may become increasingly difficult which may undermine elimination efforts. Moreover, it is worth noting that surveillance methods based solely on detection of symptomatic clinical illness are unlikely to provide accurate estimates of ongoing transmission in the Brazilian Amazon Basin –4. UnderMalaria infection imposes an antibody ‘footprint’ that will last longer than the infection itself. Anti-malarial antibody responses reflect cumulative malaria exposure in a population and may be considered an alternative tool to measure malaria transmission intensity as well as to evaluate changes in exposure . PreviouP. vivax and P. falciparum in Brazil; (b) list antigenic-used targets according to their usability; and, (c) identify knowledge gaps for malaria serological research in Brazil.There have been several studies investigating antibody responses against different malaria antigens in Brazil. However, there is a great dissimilarity between them, which may prevent a conclusive analysis to establish a comprehensive panorama of malaria epidemiology over the past decades in Brazil. Therefore, the overall aim of this study is to collate data from relevant serological surveys of anti-malarial antibodies conducted in Brazil through a systematic review strategy. Specific objectives of this study include: (a) the description of representative studies on seroprevalence of antibodies against falciparum) AND (serology OR antibody OR antibody OR immunoglobulin) AND Brazil’.The search comprised different studies designs  with reported serological results (seropositivity proportions and antibody levels) in well-defined populations. Papers included in this review were searched using Medline (via PubMed), EMBASE and LILACS  databases published up until 25 May 2016. The search was conducted using the following search terms: ‘Malaria AND  in addition to the following inclusion criteria: (1) studies must have provided a description of markers and laboratory methods used for the surveys; (2) data on the population age structure and geographical location characteristics must have been reported; and, (3) description of sample strategy and/or study design should have been reported. Studies that measured total IgG, total IgM or IgG sub-classes humoral immune responses were considered. This review included articles written in both English and Portuguese.1, IgG2, IgG3, IgG4) were analysed. Reports with insufficient data, literature reviews and studies from non-endemic areas (imported cases or outbreaks outside the Brazilian Amazon endemic area) have also been excluded.This review excluded case reports, experimental studies in animal models, studies based solely on blood bank volunteers, pregnant women, and any other particular groups that were not considered to be representative of the general population of interest. Studies were also excluded if the recruitment of individuals was based on their clinical status  or if only IgG sub-classes ; study design and sampling procedures, serological markers and methods used for measurement; parasite diagnostic methods used [microscopy or polymerase chain reaction (PCR)]; study population characteristics (age distribution and gender); seroprevalence ; parasite prevalence and transmission intensity in the study site .Two independent review authors used the inclusion and exclusion criteria to identify possible studies based on titles and abstracts. Relevant information from the selected articles was extracted and entered into a standardized Excel datasheet by the same two authors independently. Any differences were resolved on further discussion with a third author before including the data in the review. Data were extracted using a pre-defined form, which included the following information: publication data ; demographic and ecological characteristics of the study site . After removing duplicated papers, 220 articles were screened. Based on their titles and abstracts, 171 non-relevant papers were excluded and 49 full-text articles were assessed for eligibility. Six of these papers were excluded based on the inclusion and exclusion criteria . Twenty articles scored <70% when the critical appraisal tool was applied and were therefore excluded. Hence, 23 studies were included in this review for the qualitative synthesis of the published papers  used microscopic observations of thick blood smears to determine malaria infection, whereas others used PCR in addition to microscopy.The studies selected were conducted among populations living in six different states within the Brazilian Amazon Basin . Most of them analysed humoral immune responses among distinct populations living in Rondônia State. Two different studies were conducted among indigenous populations. One of them took place in four isolated indigenous tribes from the Amazon region  and the P. falciparum, 14 papers assessed P. vivax and four articles reported results on both Plasmodium species. The majority of published papers were cross-sectional surveys (82.6%) and only four cohort studies were included in this review.Five studies reported serology results on P. falciparum and P. vivax antigens were used to determine IgG-specific antibody responses among different populations in the Brazilian Amazon endemic region (Table Enzyme-linked immunosorbent assay (ELISA) alone or in combination with other methods, was the chosen serological assay used in 21 (91.3%) of the included papers. Indirect fluorescent antibody test (IFAT) was used in five studies published between 1975 and 2004. Furthermore, a considerable number of P. vivax and P. falciparum. Other recombinant antigens have also been included, such as CS, AMA1 and DBP as well as P. falciparum and P. vivax blood stages crude extract  of both P. falciparum whole parasites antigens [It is worth noting that among the 23 selected studies, eight also examined IgG subclasses responses to malarial antigens. Only four studies examined the profile of IgM responses in parallel to IgG antibodies against antigens –17 or agantigens .P. falciparum and P. vivax reported by the Brazilian Ministry of Health since 1960. The first paper on malaria sero-epidemiology included in this review was published in 1975 by Jeffery et al. [P. falciparum and P. vivax confirmed cases were diagnosed in the Brazilian endemic region. The authors ascertained malarial infection by thick blood smears and applied IFAT based on sera eluted from paper filter in populations where constant movement of people and variations in transmission intensity made the collection of reliable malariometric information particularly difficult. The overall seroprevalence  varied from 4.3 to 13.6% with a prevalence of malaria infection ranging from 0.8 to 2.3% across four surveys. Stratified seroprevalence per age group is reported only for the Cuiaba region (MT), where 2.9–7.4% of children aged 5–14 years had positive antibodies against P. falciparum or P. vivax. Of note is the fact that based on such extremely low parasite rates and relatively low IFAT positivity, the large-scale insecticide-spraying programme in Mato Grosso State was interrupted during the 1970s.The species-specific antigens used in the eligible studies were consistent with the case distribution data for During the 1980s, malaria cases increased in the Brazilian Amazon region as an effect of environmental and demographic changes during the military dictatorship regime , 20, 21.P. falciparum and P. vivax were registered in the Brazilian Basin. Indeed, two out of five of those studies, which were conducted in Acre and Pará States, used P. falciparum and P. vivax total proteins as target antigens to detect specific antibodies among endemic populations [P. falciparum blood-stage antigens as well the repeated epitopes of circumsporozoite protein [The five selected works carried out during the 1990s followed the malaria distribution trend in the late 1980s when similar number of malaria cases due to ulations , 23. Two protein , 16.P. vivax antigens   depicting low transmission rates  [P. vivax (classic and VK247 variant) and P. falciparum [P. vivax and from 0.625 to 3.3% to P falciparum at the surveyed communities. While the seroprevalence of anti-CS targeting classic P. vivax varied from 9 to 25%, the VK247 variant was less frequently observed varying from 1.2 to 10% among sampled subjects. The prevalence of antibodies to both P. falciparum and P. vivax CS proteins was higher in areas of recent migration mainly in 1987. Furthermore, an overall decline in the seroprevalence of anti-CS antibodies in 1990 compared to 1987 was reported and may reflect a significant decrease in the number of malaria cases reported by the Brazilian Ministry of Health at that time as a consequence of DDT spraying. Moreover, antibodies against CS proteins of both P. falciparum and P. vivax increased with age (p < 0.001) in the 1987 and 1990 surveys. Taken together, these results point out an association between anti-sporozoite antibodies and malaria exposure.Few cross-sectional studies aimed to characterize the antibody responses against (CS) proteins of ce <10%) , 16, 23.P. falciparum CS antibodies. A seroprevalence of 6% was revealed but no relationship observed between the frequency of anti-sporozoites (CS) and gender, time of residence or malaria infection. However, a significantly higher frequency was observed in individuals over 30 years old (p < 0.001) and in those with a history of more than nine previous malaria episodes (p < 0.001).A similar association was described in a study comprising five cross-sectional surveys between 1986 and 1989 in four different communities in Rondônia State . A totalP. vivax and P. falciparum [In agreement with the two previous published papers, data collected from cross-sectional surveys conducted between 1986 and 1988 among four indigenous Brazilian tribes in Pará State (n = 430 natives) revealed an increased anti-sporozoite seropositivity according to age for both lciparum .P. falciparum asexual blood-stage antigens [Six cross-sectional studies evaluated the IgG profiles to total antigens , 24, 26 antigens , 24, 26 antigens –16.P. falciparum were also used in cross-sectional studies aiming to define the patterns of naturally occurring antibodies in different epidemiological settings within the Amazon Basin [P. falciparum (Pf72/Hsp70-1), the 19 kDa fragment of PfMSP-1 that remains on the surface of merozoite during invasion (PfMSP-119) and the apical membrane antigen 1 of 3D7 strain (PfAMA1).Recombinant antigens of on Basin –28. SuchP. falciparum).A cross-sectional survey conducted in Candeias do Jamari (Rondônia State), a small town in a hypo-endemic area with unstable malaria transmission, selected 241 non-immune migrants using household-based randomization criteria . Blood s19. Two cross-sectional surveys enrolling 594 individuals (both children and adults) living in riverine communities along the Jau and Unini rivers (Amazonas State) evidenced a seroprevalence around 52%, which was positively correlated with age (OR 1.04 95% CI 1.019–1.06 p < 0.0001) [19 antigen (25.6%) was reported. This latter study also evaluated the humoral immune response to the apical membrane antigen 1 (AMA1) of P. falciparum. An overall prevalence of 12.7% was detected, probably reflecting the little contribution of P. falciparum to the malaria burden at the different localities studied during the years of 2006–2010.Two different studies measured naturally acquired IgG antibodies to the conserved 19-kDa C-terminal region of the merozoite surface protein 1- PfMSP-1 0.0001) . Another 0.0001) . DespiteP. vivax antigens. Among them, two works reported IgG responses to total P. vivax asexual blood stage antigens measured by IFAT [P. falciparum, the relationship between seroprevalence and age was contrasting, based on data from those two papers.A total of 13 cross-sectional studies investigated humoral responses to  by IFAT , 19. As 19 antigen. Among 13 cross-sectional studies, five were conducted aiming to measure the naturally acquired antibodies to such conserved fragment of P. vivax merozoite. Different papers have reported a positive association between anti-PvMSP-119 antibodies and malaria exposure as detailed below.Most sero-epidemiological studies in the Brazilian Amazon Basin has been focused on the PvMSP-1P. vivax infection were detected by PCR. A seroprevalence of 46.9% for PvMSP-119 was reported and the probability of a positive IgG specific response was 3.44 times higher in individuals older than 15 years in comparison to those younger. Similar results were described by Ladeia-Andrade et al. [19 was positive in 69.6%, half way through the rainy season, and 64.0% at the middle of the dry season. Age was positively correlated with seropositivity (OR 1.04 95% CI 1.019–1.06 p < 0.0001). Furthermore, very strong evidence for an association between the number of previous malaria episodes and anti-PvMSP-119 antibodies was observed among 432 subjects living in Rio Pardo, Amazonas State, in a cross-sectional study conducted in 2008 [Suarez-Mutis et al.  sampled e et al.  across t in 2008 .P. vivax merozoite antigens (PvMSP-119 and PvAMA1) in individuals living in areas of varying P. vivax endemicity in Pará State. Seroprevalence for any P. vivax antigens ranged from 19.6% (low transmission) to 77.5% (high transmission) corroborating the official Brazilian annual parasite index (API). However, low parasite prevalence (0–5%) was detected in participants from those six localities.A recent study demonstrated that serology could be used to measure and monitor transmission of malaria in the Brazilian Amazon endemic region . The aut19 and PvAMA1 was also extracted from a paper published by Sanchez-Arcila et al. [P. vivax-specific responses. A total of 279 people (14–43 years old) from a rural settlement were included. Overall seroprevalence was 74% for any antigen, 66% for PvMSP-119 and 63% for PvAMA-1. Interestingly, responses to both PvAMA1 and PvMSP-119 were lower in individuals with intestinal parasites.Data on antibodies to PvMSP-1a et al.  that evaP. vivax in communities in Rondônia State, enrolling both migrant and native populations. The first paper published in 2008 [p value = 0.02). The second paper by the same group [19, PvMSP3-α and PvMSP-9. The frequency of responders shows that IgG antibody reactivity to PvMSP-119 was 86.7% and the frequency of positive individuals for at least one of the recombinant proteins representing PvMSP-3a and PvMSP-9 sequences was 77 and 76%, respectively. Another paper conducted at those same localities in Rondônia State verified 58% of positive antibody responses to recombinant PvMSP9-RIRII and 32.5% to PvMSP9 E795-A808 protein [Other MSPs antigens have also been evaluated among Amazonian populations. Three papers included in this review used different target antigens for assessing seroprevalence of  in 2008  analysedme group  used PvMme group  sampled  protein . In agreP. vivax erythrocyte invasion [The vivax Duffy binding protein (PvDBP), a leading malaria vaccine candidate that plays a critical role in invasion , 36, hasinvasion  assessedinvasion  showing P. vivax blood-stage infections was investigated in two published papers. The first paper measures specific IgG antibodies to five recombinant fragments spanning the length of the extracellular domain of PvRBP1 [Reticulocyte binding protein 1 (PvRBP1), another protein involved in the reticulocyte-specificity of f PvRBP1 . Seropref PvRBP1  used a cP. falciparum [P. vivax [Only six cohort studies were included in this systematic review. These longitudinal studies focused on different antigens of lciparum , 25 and P. vivax , 39–41. P. falciparum, two studies were conducted in two distinct epidemiological settings. The first cohort study included in this review was published in 1994 by Ferreira and collaborators [P. falciparum) among 25 individuals. Seroprevalence for P. falciparum somatic and exo-antigens increased after the outbreak. An increase from 13 to 30% IgG positivity for exo-antigens and from 20 to 40% for somatic antigens was reported. No association between previous malaria immunity and the profile of malaria response could be drawn from this study.Regarding borators . The autP. falciparum  among 356 individuals living in a rural community in Acre State [The other cohort study analysed the prevalence of antibodies recognizing variant antigens representing the MSP-2 of re State . SeroposP. vivax, it is worth mentioning that combined results from two studies showed an evidence of association of PvMSP1 N-terminal responses and time of residence in the Brazilian endemic regions [With regard to  regions , 41. Nog regions  conducteA second paper from the same research group analysed the profile of antibodies against the N-terminal region of the PvMSP-1 in a 1-year follow-up conducted among 308 individuals living in a rural community in Amazonas State . Seropos19. The authors found a positive correlation (p < 0.005) between IgG PvMSP-1 antibodies and time of residence in the Amazon.Another cohort study included in this review was published by Bastos et al.  in 2007.The prevalence of anti-DBP antibodies was investigated in 366 people living in Ramal do Granada (Acre State) . Two croP. vivax exclusively, reflecting the epidemiological changes in Plasmodium species distribution in Brazil. Malaria cases shifted from predominantly caused by P. falciparum in the 1960s to P. vivax, the latter now being responsible for over 80% of cases in the country. Studies included in this review were highly heterogeneous and reported seroprevalence varied with the transmission risk in the area, target antigen used and target population .This systematic literature review included 23 studies reporting malaria seroprevalence data in the Brazilian Amazon endemic region. Most of the studies (60.87%) reported data on Since the Global Malaria Eradication Programme proposed by the WHO in 1955, serology has been used to measure malaria exposure and was a prominent method in early elimination attempts reported in papers published in the 1970s –44. As tP. vivax studies and another ten different targets in P. falciparum studies. Thus, a gross seroprevalence comparison across these communities with variable malaria transmission using several different targets would be inappropriate.The Brazilian Amazon region is characterized by highly variable and unstable malaria transmission. The risk of acquiring malaria varies substantially even within the same state. These different transmission patterns could explain some of the variation in seroprevalence found in the selected studies, in addition to the sensitivity of the chosen target antigen. Twelve different antigens were used in It is important to note that studies involving native indigenous populations are under-represented. Only two papers , 15 evalThere seems to be suggestive evidence that higher titres of IgG antibodies against some antigen targets  correlate with the number of years living in the endemic area and not necessarily with age. Sero-epidemiological studies conducted in Africa have shown an association with the presence of malaria antibodies and age, which has been used as surrogate marker for malaria exposure , 48. HerP. vivax [P. falciparum [Three selected heterogeneous studies conducted during the 1980s , 16, 23 Few studies attempted to investigate the association between seropositivity and months since the last malaria episode. However, the cross-sectional design adopted by most of the authors is subject to recall bias from individuals surveyed. The criteria used to ascertain previous malaria infections is not clearly defined in any of the reported cross-sectional studies, apart from the paper assessing PvCS and PfCS antigens where positivity against these targets was clearly associated with individuals reporting around ten previous malaria episodes .P. falciparum and P. vivax has been considered a potential marker for protection against malaria as well as a surrogate marker for malaria exposure in geographically diverse populations [19/PfMSP-119 and malaria exposure parameters  in cross-sectional sero-epidemiological studies [19 antigens are exclusive to each Plasmodium species, considerable homology has been reported among them [19 antigens of the four major human malaria parasites are species-specific in Zimbabwean populations [19 serological responses, an issue that remains to be established among Brazilian Amazonian populations.The 19 kDa conserved fragment of MSP-1 from both ulations , 50. Thi studies , 27–29. ong them  and, theulations . Here noP. vivax infections by measuring exposure to mosquito saliva through detection of anti-saliva antibodies or through the measurement of hypnozoite-specific signals [P. vivax has a more variable and diverse genome than P. falciparum. Thus, multi-antigenic approaches such as micro-array would allow more detailed screening of the different antigen variants that could possibly be used to distinguish relapses from new infections [It has been suggested that serological methods might be used to distinguish between relapse and new  signals . Howeverfections , 54.P. falciparum, and more recently for P. vivax, in regions of high and low transmission [P. vivax infection compared to P. falciparum, which may be due to the differing biological features related to the two species, such as the ability of P. vivax to maintain hypnozoites in the liver. Thus, a differential contribution of antibodies to NAI in P. vivax and P. falciparum infections is expected. Brazilian malaria distribution since the early 1960s and the fluctuations in proportions of slide-confirmed P. falciparum and P. vivax infections diagnosed over the last decades represent an ideal scenario to address this puzzling question. Notwithstanding, little is known about NAI in areas of less intense malaria transmission in the Amazon Basin mainly due to few cohort studies being conducted in the region.Antibodies are important mediators of naturally acquired immunity (NAI) that controls blood-stage parasitaemia, thereby reducing clinical symptoms and life-threatening complications. The role of specific antibodies is evidenced by experimental animal models and, most importantly, classical passive transfer studies in which antibodies from malaria immune adults were successfully used to treat children with severe malaria , 56. Morsmission . InteresAsymptomatic malaria has been a subject of increasing interest in the Brazilian context but is still lacking substantial data. Serology has the potential to detect not only ongoing blood-stage infections missed by conventional microscopy, but also recent infections, either symptomatic or not. Despite this potential, the use of serology for targeting asymptomatic carriers has been rarely reported in Brazil and the available data do not provide conclusive or comparative results.Sensitive diagnostic methods, such as PCR, and an appropriate follow-up period are required to identify asymptomatic malaria infection. Certainly, these requisites impose substantial costs on epidemiological surveys, which may explain the scarce number of longitudinal sero-epidemiological studies in Brazil. From all the studies that attempted to investigate the association between seropositivity and asymptomatic infection, two cohort studies suggested that PvMSP-1 N-terminal region might be a target for protective immunity , 41. TheAlthough serology has been recognized as a valid surveillance tool, the vast majority of studies in this review used it as the means to identify potential targets for the development of a vaccine against the disease. Major issues with the representativeness of the sample arise from this approach, as most of the authors used a convenient sampling method instead of structured sample size calculations and random selection of individuals. Despite the bias introduced by the sampling methods used and the focus on identifiable targets for a candidate vaccine, valuable malaria transmission information can still be retrieved from these studies.P. vivax infections. There is a gap in research to be filled by better designed, cross-sectional, epidemiological surveys with improved sampling procedures. Surveys focusing on the broader epidemiological and sero-surveillance aspects of malaria seroprevalence are needed, considering the aim of most published studies included in this review was to identify prospective malaria vaccine candidates.The review also highlights the need for longitudinal studies to better understand the role of these antibodies in malaria protection and asymptomatic 19 proteins, of both P falciparum and P. vivax, may be considered as putative biomarkers of malaria exposure in areas with variable and unstable malaria transmission in the Brazilian Amazon. However, standardization of target antigens and their relationship with malaria exposure are issues that require extra attention and need to be addressed before seroprevalence methods can be incorporated as a surveillance tool into the Brazilian malaria programme. A useful biomarker to differentiate relapses from new P. vivax infections is yet to be explored by further research. Furthermore, the proposed critical-appraisal, score-based, weighted tool developed and used in this study to assess cross-sectional studies still requires further validation.This systematic review reveals that IgG antibodies against two parasite proteins, the CS and the MSP1In conclusion, efforts will need to be made to ensure greater consistence among further sero-epidemiological studies in the Brazilian Amazon endemic region to improve the ability to compare different results that should contribute to understanding malaria transmission.


In the wake of a decline in global malaria, it is imperative to describe the epidemiology of malaria in a country to inform control policies. The purpose of this study was to describe the epidemiological and clinical profile of paediatric malaria in five epidemiological strata of malaria in Cameroon including: the Sudano-sahelian (SS) strata, the High inland plateau (HIP) strata, the South Cameroonian Equatorial forest (SCEF) strata, the High western plateau (HWP) strata, and the Coastal (C) strata.This study involved 1609 febrile children (≤15 years) recruited using reference hospitals in the five epidemiological strata. Baseline characteristics were determined; blood glucose level was measured by a glucometer, malaria parasitaemia was assessed by Giemsa microscopy, and complete blood count was performed using an automated hematology analyser. Severe malaria was assessed and categorized based on WHO criteria.p < 0.001) and in Limbe (C strata) (p < 0.001). The overall rate of severe malaria (SM) attack in this study was 29.3%; SM was significantly higher in children below 60 months (p < 0.046). Although not significant, the rate of SM was highest in Maroua (SS strata) and lowest in Limbe in the C strata. The main clinical phenotypes of SM were hyperparasitaemia, severe malaria anaemia and impaired consciousness. The majority (73.2%) of SM cases were in group 1 of the WHO classification of severe malaria (i.e. the most severe form). The malaria case-fatality rate was 5.8%; this was higher in Ngaoundere (HIP strata) (p = 0.034).An overall prevalence of 15.0% (95% CI: 13.3–16.9) for malaria was observed in this study. Malaria prevalence was significantly higher in children between 60 and 119 months (In this study, malaria prevalence decreased steadily northward, from the C strata in the South to the SS strata in the North of Cameroon, meanwhile the mortality rate associated with malaria increased in the same direction. On the contrary, the rate of severe malaria attack was similar across the different epidemiological strata. Immunoepidemiological studies will be required to shed more light on the observed trends. Malaria is a disease associated with great morbidity and mortality especially in children in sub-Saharan Africa (SSA). According to the WHO, there were 214 million cases and 438,000 deaths attributed to malaria in 2015  (Table p = 0.003). A majority of children with CM had severe malaria anaemia (SMA) compared to those without CM (85.7% vs. 42.9%). Fewer children with CM had RD compared to those without Severe malaria anaemia (SMA)In all, 19.7% 14/71) of the participants had CM (Blantyre Coma Score ≤ 2). The proportion of CM was similar in all age groups (/71 of thp = 0.046). The proportion of SMA was similar in males and females , and the proportion was also similar between the different study sites (p = 0.223) (Table 3)Respiratory distress (RD)This was the second most frequent manifestation 64.8% 46/710) that occurred in the affected children. SMA was more frequent in children <60 months 3) Table . The ove Table . 10 that op = 0.048). The proportion of RD was similar between males and females . The proportion was also observed to be similar between the different study sites (p = 0.914) (Table About 38% 27/71) of the participants presented with RD at baseline. Children with RD were more likely to be younger and <60 months p = 0.08. The pr/71 of thOther SM phenotypes at enrollment were hyperpyrexia 48 (67.6%), impaired consciousness 40 (56.3%), hypoglycemia 20 (28.2%), hyperparasitaemia 18 (25.4%), multiple convulsion 18 (25.4%), circulatory collapse 16 (22.5%), frequent vomiting 14 (19.7%), coma 14 (19.7%), jaundice 13 (18.3%), and prostration 12 (16.9%).The majority of SM cases were in group 1 (i.e. most severe form of SM) on the WHO severity index, meanwhile group 3 (i.e. the least severe form of SM) had the least number of cases Fig. .Fig. 4Dip = 0.616) or age (p = 0.377) or study site (p = 0.561) .The overall case fatality observed in this study was 5.8% (14/242). The case fatality rate were 7.8% (8/103), 3.7% (3/81), 5.1% (3/59) for age group <60, 60–119, ≥120 respectively. However no significant association was observed between fatality rate and age (p = 0.494).The case fatality rate was higher in males 8.1% (10/123) compared to females 5.9% (7/119). However no significant association was observed between fatality rate and gender (p = 0.034).The case fatality rate were 2.2% (1/46), 1.2% (1/86), 9.7% (6/62), 10.5% (2/19) and 13.8% (4/29) for HWP, C, SCEF, SS, and HIP strata respectively. A significant association was observed between the fatality rate and study site . The variation in the prevalence of malaria between the study sites could be attributed to the different levels of transmission; malaria could be described as hyperendemic in Limbe [The overall prevalence of malaria in the current study was 15.0%. This decreased steadily from the South towards the North of the country, being highest in Limbe (C strata) and lowest in Maroua (SS strata). The association between prevalence of malaria and study site was significant , and this is consistent with studies performed elsewhere [Malaria prevalence was higher in children between 60 and 119 months of age , which is consistent with studies performed elsewhere [No significant association was observed between prevalence of malaria and gender in the current study . The observation of higher GMPD in Yaounde had previously been reported by Achidi et al. [p < 0.001). High parasite density (hyperparasitaemia) is a known risk factor for severe malaria and this may explain the higher parasite density in severe malaria cases.We found an overall geometric mean parasite density (GMPD) of 22,576.9 parasites/μl. This varied considerably across the different study sites, being highest in Yaounde and lowest in Maroua  attack in the current study was 29.3%. This rate is similar to that reported in a similar study , but difThe main clinical phenotypes of SM in this study were hyperpyrexia, severe malaria anaemia, and impaired consciousness, which is in line with similar studies performed in other areas of Cameroon , 23, 56,p = 0.034), increasing from Limbe (1.2%) northwards, where it peaked in Ngaoundere (13.8%). As mentioned above, this observation may be due to the variation in the immune responses which in turn is related to the differences in their exposure to the malaria parasites. Furthermore, univariate analysis revealed that coma, hyperparasitaemia, hypoglycaemia, circulatory collapse, cerebral malaria and severe malaria anaemia were all independent predictor of death.The overall case fatality rate was 5.8%, which is comparable to the rate reported by Achidi et al. . The casThis study which is one of the first of its kind, provides an interesting insight into the epidemiology of paediatric malaria in Cameroon, taking into consideration the variations across the different epidemiological strata of malaria. These findings may contribute significantly to programmes aimed at controlling malaria in the country. However, the seasonal variation of malaria and its clinical phenotypes were not investigated as the study was performed only during the rainy season during which malaria transmission is highest. These findings may not be generalizable to reflect the overall prevalence of malaria in the target population. Furthermore, study participants were recruited using health facilities in urban centers, these findings may not necessarily reflect the situation in rural areas. Larger studies involving rural areas will therefore be needed to shed more light.The overall malaria prevalence in this study was 15.0%. Malaria prevalence was significantly higher in Limbe in the Coastal strata and in children between 60 and 119 months. Malaria prevalence was observed to decrease steadily from the Coastal strata in the South to the Sudano-sahelian strata in the North, meanwhile the mortality rate associated with malaria increased in this direction. The rate of severe malaria attack was similar across the different study sites. Children below 60 months were most at risk of severe malaria attack. Hyperpyrexia, severe malaria anaemia and impaired consciousness were observed to be the main clinical presentation of severe malaria. The clinical phenotypes of severe malaria varied considerably across age groups and epidemiological strata; severe malaria anaemia and respiratory distress were more common in children below 60 months meanwhile cerebral malaria was significantly associated with the study site. Coma, hyperparasitaemia, hypoglycaemia, circulatory collapse, cerebral malaria and severe malaria anaemia were the independent predictor of mortality associated with malaria in the current study. Larger studies will therefore be required to shed more light on the seasonal variation of malaria and its clinical phenotypes in children in the different epidemiological strata.


Out of the 299 patients, 221 patients were positive by presumptive diagnosis, 35 were positive by Rapid Diagnostic Test (RDT), and 25 were positive by microscopy. Using microscopy as the gold standard, RDT had sensitivity of 62.5% and specificity of 92.73%, whilst presumptive diagnosis had a sensitivity of 70.83% and specificity of 25.82%. The RDT recorded ROC of 0.697 with p value of 0.0001. The presumptive diagnosis recorded ROC of 0.506 with p value of 0.7304. Though none of the test methods evaluated over the gold standard achieved the WHO recommended diagnostic sensitivity and specificity, the RDT achieved an acceptable agreement with the gold standard.Differences in quality performance score had been reported for the routinely used diagnostic methods for malaria at different settings. There is therefore a need to evaluate the test performance of the routine diagnostic methods for malaria detection in Ho, a setting with no recorded quality evaluation on malaria diagnosis. The hospital-based cross-sectional study was conducted comprising 299 outpatients. Patients were first seen and presumptively diagnosed with malaria by a clinician and were referred to the laboratory for confirmation (microscopy and Rapid Diagnostic Test). The performance analysis included sensitivity, specificity, receiver operating characteristics (ROC), weighted kappa, Youden index, and  Malaria continues to be a worldwide burden despite global efforts to curb the disease . In GhanDespite an obvious need for improvement, malaria diagnosis is the most neglected area of malaria research . Prompt A purposive convenient cross-sectional study was carried out between January 2016 and April 2016 at the Ho Municipal Hospital in the Volta Region of Ghana. The study population is comprised of all outpatients who presented with signs and symptoms common to malaria infection . These patients, aged between five (5) months and eighty-five (85) years, were first seen, presumptively diagnosed with malaria by clinicians, and referred to the laboratory for confirmation (microscopy and Rapid Diagnostic Test). Participation was voluntary and patients who were excluded were those who were unwilling to participate, inpatients, patients reporting for review, and those with unrelated cause of ailment to malaria.Using the average monthly total malaria test requested (528) for two previous months (November 2015 and December 2015), a total study population of 2112 was generated for the four months study duration, using the Raosoft online sample size calculator . The recommended minimum sample of 289 participants was calculated at 95% confidence level, 5% margin of error, and a response distribution of 68% based on the average routine laboratory malaria positive test in the two previous months irrespective of the method used.Using standard phlebotomy procedure, about 2 mL of venous blood was drawn and dispensed into ethylenediaminetetraacetic acid anticoagulant (EDTA) tubes by qualified technicians working in the hospital facility. The sample was then taken to the laboratory and used for the Rapid Diagnostic Test and field microscopy testing.μl disposable capillary pipette, whole blood was drawn and transferred into the round sample well. Four (4) drops of assay diluent was added into the square assay diluent well holding the diluent bottle vertically. Reading of test was done in 15 minutes and for samples that tested negative repeated reading was done in 30 minutes. The presence of one colour band (“C” Control line) within the result window was interpreted as negative results. The presence of two colour bands (“T” Test line and “C” Control line) within the result window was interpreted as positive results. In the event where the control line fails to appear within the results window, the test was invalidated.Rapid Diagnostic Test was carried out as routinely done without any special attention given to the samples by biomedical scientist working in the municipal hospital laboratory. All Rapid Diagnostic Tests were done using Bioline SD malaria antigen Pf. manufactured by Standard Diagnostic, Inc, Korea. Assays were carried out as described by the manufacturer. In brief, all kit components and specimen were brought to room temperature prior to testing. Using a 5 μL and 2 μL of the blood sample was pipetted for the preparation of thick and thin blood films, respectively. Thin film was fixed with methanol for 5 minutes and both thin and thick film were stained with 10% Giemsa for 10 minutes. Stained slides were left to air-dry before examination using a 100x objective oil immersion light microscope. The smears were independently read by two microscopists who were blinded to the results of the RDT as well as the diagnosis made by the clinicians and between each other. Parasites were counted against 200 white blood cells (WBCs) from the thick film. The parasite density was obtained by assuming a total WBC count of 8000/mL and at least 200 fields were examined before being taken as a negative result.An amount of 6 The sensitivity and specificity of each of the three test methods were calculated by comparing to a composite reference gold standard generated from the three methods. The composite reference method was defined as a method that was positive for malaria parasites by all of the three methods  and also negative for malaria parasites by all of the three methods. This gives the method 100% hypothetical sensitivity, specificity, and positive and negative predictive values .p values, and Youden J. statistics using MedCalc Version 14.2.0.0 for Windows .Taking blood slide microscopy as the gold standard, the performance of the presumptive diagnosis method and the Rapid Diagnostic Test was evaluated to generate diagnostic accuracy summary statistics including receiver operative characteristics test, weighted kappa, Using a composite reference as gold standard which was generated from the three diagnostic methods, only 10 people were found to be truly positive for malaria and 65 people were found to be truly negative giving a hypothetical 100% sensitivity and specificity. Evaluating each of the diagnostic methods over the composite reference, RDT reported a sensitivity of 4.78% and a specificity of 72%. The Presumptive diagnosis method showed a sensitivity of 43.48% and a specificity of 23.55%, while the field microscopy method had a sensitivity of 4.55% and a specificity of 82.28%. Using field microscopy as the gold standard, RDT reported a sensitivity of 62.5% and a specificity of 92.73%, while presumptive diagnosis reported a sensitivity of 70.83% and a specificity of 25.82%. The presumptive diagnosis was found to have a low positive predictive value (7.69) against the field microscopy technique. See Comparing the two diagnostic methods to field microscopy in relation to the different age groups, RDT reported the highest sensitivity of 85.71% among ≤5 years age group and specificity of 100% among the age group of 6–18 years. Using presumptive diagnosis, the highest sensitivity of 75% was reported among the age group of 19–40 years and a specificity of 92.31% was reported for the 6–18 years age bracket. The presumptive diagnosis exhibited a low positive predictive value across the different age categorization; the exception though was found for the 6 to 18 years group. See The quality of the test method as a medical diagnostic tool was assessed using the Youden J. index on a scale of 0-1 as shown in Tables Using the interrater agreement analysis, the weighted kappa was estimated to assess how results produced by RDT and presumptive diagnosis agree with field microscopy in the diagnosis of malaria. Agreement ranged from moderate (0.457) for RDT to poor (−0.007) for presumptive diagnosis.p < 0.0001) than the presumptive diagnosis . As seen in Tables Assessing the effectiveness of the RDT and the presumptive diagnosis as methods for the diagnosis of malaria, the receiver operating characteristic (ROC) analysis was used to estimate the area under the diagnostic curve (AUC), ranging from 0.5, a worthless test, to 1, a perfect test. The RDT was shown to be a more effective test method  had a parasitic count of 1000/μl or greater. Among the false negative recording using presumptive diagnosis 14.29% was within the parasitic density of 1–100/μl and the rest (85.71%) presented with parasitic density of 1000/μl and above. See Out of the 24 patients that were reported as having positive malaria parasite by the field microscopy method, 15 and 17 were positive for RDT and presumptive diagnosis, respectively (true positive). Among the 264 patients who tested negative for RDT, 9 were positive for field microscopy . Among the 78 patients reported to be negative by presumptive diagnosis, 7 tested positive using the field microscopy technique . Among the false negative recording using the RDT 11.11% was within a parasitic count of 101–1000/Malaria is a deadly disease especially in children and, for that reason, it is important to have a prompt and accurate diagnosis of the disease . In thisDiagnosis of malaria has traditionally been based on presumptive diagnosis  and its  histidine-rich protein 2 (Pfhrp2) assay was found in children compared to adults. Nkrumah et al. [After age stratification, the highest sensitivity for the two test methods against the field microscopy technique was recorded among ≤5 years group . This agh et al.  posited h et al. . histidine-rich protein 2 (Pfhrp2) have shown a varied accuracy for malaria infection in field studies, with field microscopy taken as the gold standard [ Plasmodium falciparum densities of 100/μL and a specificity of 95% for an acceptable Rapid Diagnostic Test for malaria [Rapid Diagnostic Tests (RDTs) based onstandard , 18–20. standard , 22. How malaria . The dia malaria .Persistence of Pfhrp2 protein in circulation after parasite clearance contributes to lower specificity level of RDT , 19, 21.μL  as seen in Youden index was carried out to measure the medical usefulness of the diagnostic methods for malaria detection. RDT proved to be a useful diagnostic test (Youden: 0.395) as compared to presumptive diagnosis (Youden: 0.013). The performance of the RDT agrees with the findings of Samadoulougou et al.  in BurkiBased on the results of this study, none of the test methods evaluated over the gold standard (field microscopy technique) achieved the WHO recommended diagnostic sensitivity and specificity. However, the RDT achieved an acceptable agreement with the gold standard. Factors such as medication and age also played a role in influencing the test performance of the various diagnostic methods. Diagnosis of malaria by field microscopy should still be the gold standard although it requires a level of expertise. In settings where microscopy is not available, however, RDTs must be preferred as a confirmation of presumptive diagnosis.


Oxylipins and endocannabinoids are low molecular weight bioactive lipids that are crucial for initiation and resolution of inflammation during microbial infections. Metabolic complications in malaria are recognized contributors to severe and fatal malaria, but the impact of malaria infection on the production of small lipid derived signalling molecules is unknown. Knowledge of immunoregulatory patterns of these molecules in malaria is of great value for better understanding of the disease and improvement of treatment regimes, since the action of these classes of molecules is directly connected to the inflammatory response of the organism.®) and univariate approaches (receiver operating characteristic (ROC) curves, t tests, correlation analysis).Detection of oxylipins and endocannabinoids from plasma samples from forty children with uncomplicated and severe malaria as well as twenty controls was done after solid phase extraction followed by chromatography mass spectrometry analysis. The stable isotope dilution method was used for compound quantification. Data analysis was done with multivariate , orthogonal partial least squares discriminant analysis (OPLS-DA2) in significantly lower levels and four endocannabinoids  at significantly higher levels in infected individuals as compared to controls according to t test analysis with Bonferroni correction. Three oxylipins  were higher in severe compared to uncomplicated malaria cases according to the results from multivariate analysis. Observed changes in oxylipin levels can be connected to activation of cytochrome P450 (CYP) and 5-lipoxygenase (5-LOX) metabolic pathways in malaria infected individuals compared to controls, and related to increased levels of all linoleic acid oxylipins in severe patients compared to uncomplicated ones. The endocannabinoids were extremely responsive to malaria infection with majority of this class of molecules found at higher levels in infected individuals compared to controls.Forty different oxylipin and thirteen endocannabinoid metabolites were detected in the studied samples, with one oxylipin  contains supplementary material, which is available to authorized users. Plasmodium falciparum malaria, is a complex syndrome characterized by the sequestration of parasitized erythrocytes in the peripheral vasculature and a profound imbalance between pro-and anti-inflammatory responses. The acute phase response carries many common denominators with sepsis, systemic inflammatory response syndrome and in critical illness [Malaria still poses a strenuous burden on the health systems in low resource countries. The most dangerous form of the disease, severe  illness , 2. Howe illness .Metabolic complications of malaria are increasingly recognized as contributors to severe and fatal malaria . AlthougMetabolomics, the study of small molecules, can be the most useful approach to study disease from the clinical perspective. All aforementioned methodologies, including metabolomics, are not based on any pre-assumptions and preconceptions. As such, they have the advantage of unbiased identification of disease-related signals. Initial differences in metabolite levels may be diagnostic of disease, or determinate its severity or stage . As metaMost of the literature reports regarding chemical analysis of plasma from humans infected with malaria deal with global screening of metabolites or lipids –11. It wSmall lipid mediators are found in human tissues in very low amounts (pM–nM) and hence are usually not detected with global profiling methods. An analysis of these molecules needs to be performed not only after dedicated sample clean-up, but also with specific targeted approaches. Liquid chromatography with triple quadrupe mass detector is the method most suited for analysis of very low abundant metabolites in complex matrices and hence optimal for investigation of pro- and anti-inflammatory small molecules .In this work a number of physiologically relevant oxylipins and endocannabinoids were quantified in plasma from malaria patients and controls with liquid chromatography tandem mass spectrometry (LC–MS/MS) approach. Investigated compounds were previously detected in plasma and belong to the main small lipid mediator synthesis pathways Fig.  with knoA total of 690 patients between 6 months and 6 years of age were enrolled from January 2011 to September 2013 at Nyagatare Hospital in Nyagatare district, Kiziguro Hospital and Ngarama Hospital in Gatsibo district or health centres in the catchment areas of the 3 hospitals in the Eastern Province of Rwanda. The patients were assessed on site by the attending paediatrician/study medical officer, and biometric and clinical parameters were recorded, as listed in Additional files Representative individuals were chosen from the 690 samples, based on clinical information. For the lipidomics analysis, we selected 20 samples from each group of diagnostic categories , 10 of each gender using a two factor full factorial design applied to principal component analysis (PCA) plots of two-component models on all available samples with clinical data as X variables , Additi. Because4 and 12(13)-EPOME-d4, 25 ng/mL 9-HODE-d4, PGE2-d4 and TXB2-d4, 800 ng/mL 2-AG-d8, 40 ng/mL PGF2α-EA-d4 and PGE2-EA-d4, 20 ng/mL AEA-d8, OEA-d4, SEA-d3 and PEA-d4 as well as 10 µL antioxidant solution (0.2 mg/mL BHT/EDTA in methanol/water (1:1)). After applying the sample, IS, and antioxidant solution, the SPE cartridge was washed with 5% MeOH, 0.1% acetic acid in water, dried under high vacuum and eluted with 2 mL methanol, 3 mL acetonitrile and 1 mL ethyl acetate into polypropylene tubes containing 6 µL of a glycerol solution (30% in methanol). Eluates were evaporated using a MiniVac system , reconstituted in 100 μL of MeOH, spiked with 10 µL recovery standard (25 ng/mL CUDA), transferred to vials and analysed by LC–MS/MS.Plasma was prepared on site in Rwanda and snap frozen on site in liquid nitrogen. It was stored and transported in liquid nitrogen to Sweden, thawed once for aliquoting and frozen again at −80 °C. A previously reported solid phase extraction (SPE) protocol  was modi18 column  with an online filter, and injection volume was 10 µL for each run. The eluents in the mobile phase consisted of (A) 0.1% acetic acid in MilliQ water and (B) acetonitrile:isopropanol (90:10). For oxylipin separation the following gradient was employed: 0.0–3.5 min 10-35% B, 3.5–5.5 min 35–40% B, 5.5–7.0 min 40–42% B, 7.0–9.0 min 42–50% B, 9.0–15.0 min 50–65% B, 15.0–17.0 min 65–75% B, 17.0–18.5 min 75–85% B, 18.5–19.5 min 85–95% B, 19.5–21 min 95–10% B, 21.0–25.0 min 10% B [An LC–MS/MS instrument, the Agilent Ultra-Performance (UP)LC system (Infinity 1290) coupled to an Agilent 6490 Triple Quadrupole with an electrospray ionization source (ESI) equipped with the iFunnel Technology , was used with separate injections for subsequent ionization in positive (endocannabinoid) and negative (oxylipin) mode. Chromatographic separation was done on a Waters BEH Cin 10% B . The sepThe electrospray ionization and mass analysis conditions were: capillary and nozzle voltage at 4000 and 1500 V, drying gas temperature 230 °C with a gas flow of 15 L/min, sheath gas temperature 400 °C with a gas flow of 11 L/min, the nebulizer gas flow was 35 psi, and iFunnel high and low pressure RF were set at 90 and 60 V (negative mode) and 150 and 60 V (positive mode). Dynamic multiple reaction monitoring (MRM) mode was used with fixed time windows (retention time ± 2 min) to profile two transitions per compound . The dynamic MRM option was performed for all compounds with optimized transitions and collision energies. The MassHunter Workstation software was used for instrument control and for manual integration of all peaks.8, SEA-d3, PEA-d4, OEA-d4 and 2-AG-d8), and eight for oxylipin quantification -EpOME-d4, 9-HODE-d4, PGE2-d4, PGD2-d4, 5-HETE-d8, 20-HETE-d6 and TXB2-d4). For each native compound, a suitable IS was selected based on structural similarities , m is equal to the slope of the curve, x corresponds to the on column concentration of the analyte and b is the y-interception of the calibration curve.The stable isotope dilution method was used to quantify each class of bioactive lipids (oxylipins and endocannabinoids together with related lipids). Two types of internal standard were used: (i) for quantification purposes, deuterated IS was added before extraction, and (ii) for the purpose of monitoring the loss of IS, the recovery standard CUDA was added after extraction . Five ISThe SIMCA software version 14.0 from MKS Umetrics AB  was used for all data modelling. All data was log-transformed, column mean centred and divided by its standard deviation, i.e. scaled to unit variance. principal component analysis (PCA) was used to investigate overall variability within the data and OPLS-DA  to model1α was detected only in one control, two uncomplicated and two severe cases and 5(S)6(S)-LXA4 was detected in one control, two uncomplicated and three severe samples. Both compounds were hence not suitable for obtaining statistically relevant information and were removed from further data processing. Resolvin D1 displayed three values in uncomplicated malaria group, five in controls and seven in severe cases, therefore, this metabolite was also not suitable for univariate statistics, but was kept in the OPLS-DA models. Because the levels of the majority of the compounds did not follow Gaussian distribution  in any of the groups studied, further analysis focused on log-transformed data. The transformed data followed normal distribution in the majority of cases and hence parametric tests could be used to study this data. The exception was 9-HODE in the control group that was further analysed by non-parametric tests.Forty different oxylipin metabolites were detected in the studied samples with levels in the range of 0–98.7 nM, with 9,10-DiHOME and 12,13-DiHOME being the most abundant. The distribution of concentration levels in each of the studied groups of samples  is presented in Additional file Principal component analysis performed on the log-transformed data was used to get an overview of the data from the 60 samples and 38 oxylipin species. On the PCA score plot Fig.  partial P(corr) values from the studied models together with metabolites significant according to the jack-knifing are listed in Table 2, 5,6-DHET, 20-HETE, 15-HETrE, 12(13)-EpOME and 9(10)-EpOME. The same compounds (except 15-HETrE) plus eight additional  were significantly different between severe malaria and controls. One compound, TXB2, passed Bonferroni correction (threshold of 0.00125) for severe-controls and uncomplicated-controls comparison. Three compounds were significantly different between severe and uncomplicated cases: 13-HODE, 9-HODE and 13-oxo-ODE, with the last showing no difference between infected and not infected individuals. A group distribution of significant compounds is presented in Additional file According to the univariate statistics Table , six comCorrelation of oxylipin levels to the available clinical and personal descriptors of the patients’ revealed that twenty-one oxylipins were significantly positively correlated with temperature in controls, but not in infected individuals ; five were associated with severe versus control -EpOME) and one associated with uncomplicated versus control (15-HETrE) separation.Receiver-operator characteristic (ROC) curves were generated for each metabolite to test its diagnostic performance for the classification of samples into one of two groups: uncomplicated versus controls, severe versus control and severe versus uncomplicated cases. The values of the area under the curve (AUC) for all oxylipin metabolites are summarized in Additional file 2) was the only compound that survived the Bonferroni cut off value (0.00125) for both severe versus controls and uncomplicated versus controls comparisons with corresponding ROC curves presented in Fig. 2 had a Youden score < 923.4 pM (uncomplicated vs controls) and <609.5 pM (severe vs controls). Indeed, 18/20 of the uncomplicated cases had plasma levels lower than 923.4 pM and only 4/20 of the controls were lower than this value. For the severe cases 15/21 had levels lower than 609.5 pM whereas 4/20 of the controls had a lower value.Thromboxane B2 (TXBue 0.0012 for both2α-EA (present in 89% of the samples) all metabolites were detected in all samples above limit of quantification (LOQ). Given that all metabolites failed to pass D’Agostini and Pearson omnibus normality test, log transformation was done resulting in normally distributed data, allowing the use of parametric basal statistical tests. The exception was DHEA, which was further analysed by non-parametric tests.A total of 15 eCB were investigated and 13 were detected at levels in the range of 0.004–253 nM with 2-arachidonyl glycerol (2-AG), palmitoyl ethanolamide (PEA) and oleoyl ethanolamine (OEA) being the most abundant. The median and range of values for all detected metabolites are given in Additional file Principal component analysis (PCA) on the log-transformed data was used to get an overview of the data from the 60 samples and 12 endocannabinoid species. On the PCA score plot . SUS plot analysis of p(corr) vectors from uncomplicated-controls and severe-controls models revealed high correlation of their endocannabinoid profiles  values from the studied OPLS-DA models together with metabolites significant according to the jack-knifing are listed in Table Comparisons were made separately between controls and patients with different infection stages—uncomplicated and severe. Seven out of thirteen studied endocannabinoids were at significantly higher levels in infected individuals compared to controls; these were: 2-AG, OEA, PEA, DEA, EPEA, DHEA and POEA. From the significant metabolites, four  passed Bonferroni correction (threshold of 0.0042) both for control versus uncomplicated and control versus severe and two metabolites (2-AG and DHEA) passed the correction only for severe-controls comparison. There were no differences in endocannabinoid levels between severe and uncomplicated malaria cases. Group distribution of significant compounds is presented in Additional file Two compounds significantly correlated with parasitaemia scores: oleoyl ethanolamide  and eicosapentaenoyl ethanolamide . The last correlation was however driven by one sample with extreme parasitaemia score and it lost significance after removal of this sample  were common to uncomplicated versus control and severe versus control groups. POEA significance was specific to uncomplicated versus control group.The significance of the ROC curves statistics for the studied endocannabinoids including the compounds that survived Bonferroni cut-off value (0.0042) for the differences between both severe versus controls  and uncomplicated versus controls  is presented at Fig. Oxylipin and endocannabinoid levels were analysed using two statistical approaches; univariate, in order to evaluate the statistical performance of single metabolites and multivariate in order to visualize the correlation patterns of the data and obtain compound profiles characterizing the disease that could be put into biochemical context. Whereas analysis of biomarkers is more relevant in the view of disease diagnosis and prognosis, analysis of perturbed metabolic pathways can improve our understanding of the disease itself and in the future treatment improvement.2) that was differentiating infected versus controls groups. More oxylipins were significant according to t-test without multisampling correction though, including three oxylipins  that were at significantly higher levels in severe compared to uncomplicated malaria cases. Bonferroni correction is very stringent and it has been argued if its application does not result in too low a cut-off when looking for potential biomarkers in profiling studies [The diagnostic properties of the studied molecules can be evaluated by p values and ROC curve analysis. In presented study, the endocannabinoid group was found to be more useful for discrimination between infected versus controls, with a total of four significant metabolites according to the t test with Bonferroni correction . Only one significant compound was found in the oxylipin pool . This means that in our study it was the most significant compound among the studied oxylipins able to differentiate between malaria patients and controls. It was present at lower levels both in severe and uncomplicated patients, a fact that contradicts previous work conducted in a Plasmodium berghei hamster model [2 is a non-enzymatic decomposition product of the unstable TXA2. It is produced by activated platelets and has prothrombotic properties. The role of platelets in immune and inflammatory cells was recently reviewed by Morell et al. [P. vivax [2 may indeed reflect altered platelet function during malaria infection.As mentioned above, TXBer model . TXB2 isl et al.  and blool et al. , 47. AltP. vivax . EvidencP. vivax –51. ProtP. vivax . In thisThe 5-lipoxygenase (5-LOX) pathway is the major source of leukotrienes (LTs), released mainly from myeloid cells. Leukotrienes are potent pro-inflammatory and immunoregulatory lipid messengers that play a central role in immune responses and tissue homeostasis , 53. TheThe CYP pathway comprises a diverse array of membrane bound haem-iron containing enzymes that mainly are active in the liver . Action 12/15-Lipoxygenase (12/15-LOX) plays a central role in the ‘‘class switch’’ of eicosanoid mediator biosynthesis from leukotrienes to lipoxins and resolvins, initiating the active resolution of inflammation . In presIt is important to mention that some of the molecules included in this study can be also formed by non-enzymatic action of haemozoin, as presented by Schwarzer et al. , who shoRather unexpectedly, we found that endocannabinoids were remarkably responsive to malarial infection with all compounds except NAGLy being in higher levels in severe malaria patients compared to controls and six out these at significance levels that passed Bonferroni correction.2, dampens the inflammatory response during the early phase of sepsis. This may be beneficial in order to minimize tissue damage. Inhibition of endocannabinoid system at later stages of sepsis upregulates the inflammatory response and may help to reduce mortality by eliminating invading pathogens.A number of reports have indicated that cannabinoids suppress the antibody response of humans and animals , 65, witThis work presents evaluation of the oxylipin and endocannabinoid changes in plasma from children with malaria in comparison to healthy controls. A significant response of the endocannabinoid system to the malaria infection was observed, with two compounds from this group that could be correlated to the amount of parasite in the blood. Changes in activity of oxylipin synthesis pathways in infected individuals were also observed. Presented results point to the inhibition of COX pathway and activation of the 5-LOX in malaria infection that could be connected to dysregulation of immunological balance and subsequent negative effects for the patients. An inability of the infected patient to suppress inflammatory response was supported by the inhibition of 12/15-LOX pathway. The limitation of the study is the relatively small sample size per group  and the fact that few patients had concomitant illnesses, which could have influence on the levels of investigated metabolites. Presented results provide first step towards understanding of the role of small lipid derived mediators in malaria infection and point at the possibility for using oxylipin and endocannabinoid synthesis pathways as targets in treatment of malaria.Additional file 1. Clinical information for the control patients included in the study.Additional file 2. Clinical information for the uncomplicated malaria patients included in the study.Additional file 3. Clinical information for the severe malaria patients included in the study.Additional file 4. Patient selection procedure.Additional file 5. Native standards and corresponding internal standards used in the study.Additional file 6. Standard concentrations used for calibration curves of endocannabinoids and related lipids.Additional file 7. Standard concentrations (pg/mL) used for calibration curves of oxylipins.Additional file 8. Parameters of models for oxylipins discussed in the study.Additional file 9. Parameters of models for endocannabinoids discussed in the study.Additional file 10. Oxylipin levels (pM) in the blood serum samples form controls, uncomplicated malaria and severe malaria patients.Additional file 11. OPLS-DA correlation loadings from the uncomplicated versus controls and severe versus controls models.Additional file 12. Scatter plots for oxylipins that showed significant p-values in between group comparisons.Additional file 13. Correlation of oxylipin levels with temperature.Additional file 14. ROC curves results for oxylipins.Additional file 15. Endocannabinoids levels (pM) in the blood serum samples from controls, uncomplicated malaria and severe malaria patients.Additional file 16. OPLS-DA correlation loadings from the uncomplicated versus controls and severe versus controls models.Additional file 17. Scatter plots for endocannabinoids that showed significant p-values in between group comparisons.Additional file 18. Correlation of OEA and EPEA with parasitaemia values.Additional file 19. ROC curves results for endocannabinoids.


Plasmodium species. Recently, it has been considered an alternative target when designing novel antimalarial vaccines against Plasmodium falciparum. However, the potential of Plasmodium vivax CelTOS as a vaccine target is yet to be explored. This study evaluated the naturally acquired immune response against a recombinant P. vivax CelTOS (PvCelTOS) (IgG and IgG subclass) in 528 individuals from Brazilian Amazon, as well as the screening of B-cell epitopes in silico and peptide assays to associate the breadth of antibody responses of those individuals with exposition and/or protection correlates. We show that PvCelTOS is naturally immunogenic in Amazon inhabitants with 94 individuals (17.8%) showing specific IgG antibodies against the recombinant protein. Among responders, the IgG reactivity indexes (RIs) presented a direct correlation with the number of previous malaria episodes  and inverse correlation with the time elapsed from the last malaria episode . Interestingly, high responders to PvCelTOS (RI > 2) presented higher number of previous malaria episodes, frequency of recent malaria episodes, and ratio of cytophilic/non-cytophilic antibodies than low responders (RI < 2) and non-responders (RI < 1). Moreover, a high prevalence of the cytophilic antibody IgG1 over all other IgG subclasses (p < 0.0001) was observed. B-cell epitope mapping revealed five immunogenic regions in PvCelTOS, but no associations between the specific IgG response to peptides and exposure/protection parameters were found. However, the epitope (PvCelTOSI136-E143) was validated as a main linear B-cell epitope, as 92% of IgG responders to PvCelTOS were also responders to this peptide sequence. This study describes for the first time the natural immunogenicity of PvCelTOS in Amazon individuals and identifies immunogenic regions in a full-length protein. The IgG magnitude was mainly composed of cytophilic antibodies (IgG1) and associated with recent malaria episodes. The data presented in this paper add further evidence to consider PvCelTOS as a vaccine candidate.The cell-traversal protein for ookinetes and sporozoites (CelTOS), a highly conserved antigen involved in sporozoite motility, plays an important role in the traversal of host cells during the preerythrocytic stage of  Plasmodium, being responsible for nearly 438,000 deaths and 150–300 million new infections in 2015 , conferred partial protection in Phase III trials and fell short of community-established vaccine efficacy goals  was naturally recognized by acquired antibodies in exposed populations (P. berghei and inhibit sporozoite motility and invasion of hepatocytes in vitro (P. vivax CelTOS (PvCelTOS) has remained limited. Only recently, a study reported PvCelTOS as naturally immunogenic in infected individuals from Western Thailand. Our group, investigating the genetic diversity of genes encoding PvCelTOS in field isolates from five different regions of the Amazon forest, reveals a high-conserved profile. Together, both findings support the potential of PvCelTOS as an interesting target on P. vivax sporozoite surface, but further studies are still necessary to consolidate this protein as an alternative in future multitarget vaccines. Therefore, the present study aimed at evaluating the naturally acquired humoral immune response against PvCelTOS in exposed populations from Brazilian Amazon, determining the antibody subclass profile, identifying its B-cell epitopes and verifying the existence of associations between the specific IgG and subclass response against PvCelTOS and epidemiological data that can reflect the exposition and/or protection degree.In this scenario, cell-traversal protein of arasites . This searasites , 30. Theell pass . Interesulations , able toin vitro . However2 and a population of about 22,000 people, who live in rural areas inside the forests. Transmission of malaria in the Amazon occurs throughout the whole year, with seasonal fluctuations with maximum transmission occurring during the dry season from May to October and prevalence of infections by P. vivax, responsible for more than 85% of reported malaria cases.A cross-sectional cohort study was conducted involving 528 individuals from Rio Preto da Eva (2°50′50″S/59°56′28″W), located north of the Amazon River and 80 km distant from Manaus, the capital of Amazon state. This city has an area of 6,000 kmSamples and survey data were collected from November 2013 to March 2015. In addition, we also included, as control subjects, 10 naive individuals living in Manaus, and with no reported previous malaria episodes. Written informed consent was obtained from all adult donors or from parents of donors in the case of children. The study was reviewed and approved by the Fundação Oswaldo Cruz Ethical Committee and the National Ethical Committee of Brazil.In order to evaluate the possible influence of epidemiological factors on humoral immunity against PvCelTOS, all donors were interviewed upon informed consent prior to blood collection. The survey included questions related to personal exposure to malaria, such as years of residence in the endemic area, recorded individual and family previous malaria episodes, use of malaria prophylaxis, presence/absence of symptoms, and personal knowledge of malaria transmission. All epidemiological data were stored in Epi-Info for subsequent analysis .g, 10 min). Plasma samples were stored at −20°C and transported to our laboratory. Thin and thick blood smears of all donors were examined for malaria parasites. Parasitological evaluations were done by examination of 200 fields at 1,000× magnification under oil-immersion and a research expert in malaria diagnosis examined all slides. Donors positive for P. vivax and/or P. falciparum at the time of blood collection were subsequently treated using the chemotherapeutic regimen recommended by the Brazilian Ministry of Health.Venous peripheral blood was drawn into heparinized tubes and plasma collected after centrifugation  was cloned in the expression vector pHLsec, which is flanked by the chicken β-actin/rabbit β-globin hybrid promoter with a signal secretion sequence and a Lys-His6 tag. The protein was expressed upon transient transfection in HEK-293T cells with endotoxin-free plasmids in roller bottles . The secreted protein was purified from the supernatant by immobilized Ni Sepharose affinity chromatography. The presence of proteins in the elution samples was confirmed using 6xHis epitope tag antibody [horseradish peroxidase (HRP) conjugate] in a Western blot. The sample was concentrated using an Amicon Ultra centrifugal filter system (Life Technologies) until reaching 10 ml of final volume. Contaminant proteins and salts were removed from the concentrate by size exclusion purification (SEC) using Superdex medium in the column. Protein concentration after recovery was tested using a Bradford protein assay, and purity was assessed by silver staining and by Western blotting.As previously described , the P. o-phenylenediamine and hydrogen peroxide. Optical density was identified at 492 nm using a SpectraMax 250 ELISA reader . The results for total IgG were expressed as reactivity indexes (RIs), which were calculated by the mean optical density of an individual’s tested sample divided by the mean optical density of 10 non-exposed control individuals’ samples plus 3 standard deviations. Subjects were scored as responders to PvCelTOS if the RI of IgG against the recombinant protein was higher than 1. Additionally, the RIs of IgG subclasses were evaluated on responders individuals by a similar method, using peroxidase-conjugated goat antihuman IgG1, IgG2, IgG3, and IgG4 .Anti-PvCelTOS specific antibodies were evaluated on plasma samples from 528 exposed individuals from Brazilian Amazon and 10 healthy individuals, who had no reported malaria episodes, using enzyme-linked immunosorbent assay (ELISA), essentially as previously described , 34. BriThe prediction of linear B-cell epitopes was carried out using the program BepiPred , which iThe Emini surface accessibility (ESA) was used to evaluate the probability of predicted linear B-cell epitopes to be exposed on the surface of the protein. This approach calculates the surface accessibility of hexapeptides and values greater than 1.0 indicate an increased probability of being found on the surface . Sequenco-phenylenediamine and hydrogen peroxide. The absorbance was read at 492 nm using an ELISA reader . The results for total IgG were expressed as RIs, which were calculated by the mean optical density of the tested samples plus 3 standard deviations of pools of control individuals. Subjects were scored as positive if the RI was higher than 1.A peptide library of 32 PvCelTOS synthetic 15-mer peptides overlapping by nine amino acids  was synthesized. To evaluate the specific response to each peptide, the peptide array was performed using MaxiSorp 96-well plates  coated with PBS containing 5 µg/ml of each peptide in duplicates. After overnight incubation at 4°C, the plates were washed with PBS and blocked with PBS-Tween containing 5% non-fat dry milk (PBS-Tween-M) for 1 h at 37°C. Individual plasma samples were diluted 1:100 with PBS-Tween-M and added in duplicate wells for each sequence and the plates incubated at room temperature for 1 h. After three washings with PBS-Tween, bound antibodies were detected with peroxidase-conjugated goat antihuman IgG  followed by addition of Molecular dynamics simulations were carried out using GROMACS 5.1.2  softwarep value <0.05 was considered significant.All statistical analyzes were carried out using Prism 5.0 for Windows . The one-sample Kolmogorov–Smirnoff test was used to determine whether a variable was normally distributed. The Mann–Whitney test was used to compare RIs of IgG against recombinant PvCelTOS between studied groups. Differences in proportions of the RI of IgG subclasses and epidemiological parameters were evaluated by Fisher’s exact test and associations between antibody responses and epidemiological data were determined by Spearman rank test. A two-sided 2 = 5.761, p < 0.0164). Regarding the previous personal history of malaria, only seven individuals reported no malaria episode (1.3%). Among those who remembered the Plasmodium species, the majority (29.9%) reported infections by P. falciparum and P. vivax. The number of past malaria episodes also varied greatly among donors, ranging from 0 to 50 (mean = 7.74 ± 16.5). Finally, the time elapsed since the last malaria episode ranged from 0 to 480 months (mean = 71.7 ± 77.9). Interestingly, a correlation trend was observed between the time of residence in the endemic area and the number of previous malaria infections . Collectively, the epidemiological inquiry indicated that the studied population had different degrees of exposure and/or immunity.Most studied individuals were adults and naturally exposed to malaria infection throughout the years Table . Age ranp > 0.05).To test if the PvCelTOS is a target for naturally acquired antibodies in Amazon individuals, we first assessed the IgG reactivity profile against the recombinant protein. The plasma samples collected from the 528 individuals living in the endemic area reveal a low frequency of responders to PvCelTOS, since only 17.8% of the studied population  presented specific IgG antibodies against the protein. Interestingly, the epidemiological data were similar between responders and NRs against this protein , reflecting a wide spectrum in magnitude of naturally acquired IgG response. The IgG subclass profile was marked by IgG1, the most prevalent subclass, present in 65.96% of responders, and with major RI  compared to IgG2 , IgG3 , and IgG4  . Moreover, despite no statistical differences could be observed on the time elapsed from the last malaria episode (p = 0.15 in ANOVA test), the frequency of individuals who reported recent episodes of malaria was higher in HR (41.6%) than LR  and NR . Moreover, the proportion of RIs of cytophilic over non-cytophilic antibodies (IgG1 + IgG3/IgG2 + IgG4) presented direct correlation with RI of IgG of responder individuals , suggesting that higher RI could be associated with a cytophilic profile of humoral response against PvCelTOS. Interestingly, although the proportion of individuals with cytophilic profile was similar in both groups, HR and LR , the ratio of (cytophilic/non-cytophilic) antibodies was significantly higher in HR than LR (p = 0.0076) .Four B-cell linear epitopes were predicted I136-E143 and PvCelTOSK166-S191) were present  in peptides confirmed as naturally immunogenic. Interestingly, peptides I133-G147 and E182-D196 were recognized by IgG specific antibodies of responders to PvCelTOS in frequencies higher than 50%  and presented median of RI higher than 1 . In addition, peptides P127-V141, P139-V153, and L181-L195 were located besides the most immunogenic peptides and presented overlapped sequences, which were also recognized by IgG antibodies in moderate frequencies. Peptide I133-G147 (ASTIKPPRVSEDAYF) presented the highest IgG RI (p < 0.0001 by ANOVA test) and the highest frequency of recognition (92%) compared to all other peptides. While it contains the entire sequence of predicted epitope PvCelTOS136-143, peptides P127-V141 and P139-V153, which contain only the partial sequence of the predicted epitope, presented minor frequencies of recognition . The peptides L181-L195 and 186-196 were both partially inserted in the predicted linear epitope PvCelTOS166-191 and could be the immune dominant sequence of this longer predicted epitope. These data supported the prediction of linear B-cell epitopes PvCelTOSI136-E146 and PvCelTOSK166-S191. Conversely, peptides N13-L27, S19-V33, and S97-A111 also presented frequency of recognition about 40% . After the confirmation of five immunogenic regions and two immunodominant epitopes in PvCelTOS, we also compared the RI and frequencies between HR and LR for PvCelTOS. However, no differences were found.In order to validate the prediction data and identify possible non-predicted immunogenic regions of PvCelTOS, plasma from IgG responders to PvCelTOS was tested against 32 overlapping peptides corresponding to the complete amino acid sequence. First, 10 peptides  were broadly recognized by responders to PvCelTOS  presented a good combination of BepiPred and ESA score. The molecular dynamics and electrostatic potential surface of PvCelTOS indicate regions P127-V153, N13-V33, and L181-D186 as more flexible than E73-K93 and S97-A111  has been considered a potential novel alternative for a vaccine against malaria  in different frequencies and RIs. Moreover, the main linear epitope (ASTIKPPRVSEDAYF) presented highest IgG RI and frequency compared to all other naturally recognized peptides, suggesting that the majority of naturally acquired antibodies against PvCelTOS are directed to the C-terminal region. Moreover, T cell responses to PvCelTOS may also help to determine the immunodominant repertoire in individuals living in malaria-endemic regions, which could also supply information for the development of a vaccine for PvCelTOS. In humans, PfCelTOS derivate peptides elicited proliferative and IFN-γ responses in ex vivo ELISPOT assays using peripheral blood mononuclear cells from naturally exposed individuals living in Ghana (Information at the amino acid level about the epitopes of proteins recognized by antibodies is important for their use as biological tools and for understanding general molecular recognition events . In thisin Ghana .Plasmodium spp., Cytauxzoon, Theileria, and Babesia and considered essential to cell infection, traversal, and membrane disruption (Recently, CelTOS was demonstrated as highly conserved protein across several large groups of apicomplexan parasites including sruption . Despitesruption . These oJL-J did study designing, performed experiments, data analysis, manuscript preparation, and manuscript review. RR-d-S did study designing, performed experiments, data analysis, and manuscript preparation. IS performed experiments. CL-C did recombinant protein expression and manuscript review. JM did molecular dynamics and bioinformatics and manuscript review. DP-d-S performed collection of blood and epidemiological data. AF did fieldwork support. AT performed collection of blood and epidemiological data and diagnosis. FP did fieldwork support. LC performed experiments. LP-R did data analysis and manuscript review. AR-S did recombinant protein expression, data analysis, and manuscript review. DB did study designing, fieldwork support, manuscript review, and data analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.