Over the last decades, malaria has declined substantially in The Gambia but its transmission has not been interrupted. In order to better target control interventions, it is essential to understand the dynamics of residual transmission. This prospective cohort study was conducted between June 2013 and April 2014 in six pairs of villages across The Gambia. Blood samples were collected monthly during the transmission season (June-December) from all residents aged ≥6 months  and then in April (dry season). Entomological data were collected monthly throughout the malaria transmission season. Ownership of Long-Lasting Insecticidal Nets was 71.5% (2766/3869). Incidence of malaria infection and clinical disease varied significantly across the country, with the highest values in eastern (1.7/PYAR) than in central (0.2 /PYAR) and western (0.1/PYAR) Gambia. Malaria infection at the beginning of the transmission season was significantly higher in individuals who slept outdoors  and in those who had travelled outside the village . Sub-patent infections were more common in older children  and adults  than in younger children. The risk of clinical malaria was significantly higher in households with at least one infected individual at the beginning of the transmission season . Vector parity was significantly higher in the eastern part of the country, both in the south  and the north bank , than in the western region , indicating higher vector survival. There is still significant residual malaria transmission across The Gambia, particularly in the eastern region. Additional interventions able to target vectors escaping Long-Lasting Insecticidal Nets and indoor residual spraying are needed to achieve malaria elimination. Over the last 15 years, the burden of malaria has decreased substantially worldwide, sub-Saharan Africa included, thanks to the scale up of standard control interventions such as prompt diagnosis and treatment, long-lasting insecticidal nets (LLINs), and indoor residual spraying (IRS) , 3. ThesAn.(Anopheles) gambiae complex (sensu lato); An. arabiensis is found mainly in the eastern part of the country, An. melas, a brackish water species, in the central and western regions, and An. gambiae s.s. (sensu stricto) across the whole country [An. arabiensis is predominantly outdoor biting and may contribute to maintaining outdoor transmission. Indeed, for individuals protected by LLINs, a small but important portion of vector feeding activity occurring at dawn and dusk accounts for approximately half of all transmission exposure to residual vector populations [In The Gambia, the observed malaria heterogeneity, i.e. increasing transmission from west to east, has been partly attributed to the variation in the distribution of the vector  country . An. araulations .As transmission is markedly seasonal, it is assumed that malaria-infected but otherwise healthy individuals, i.e. without any clinical symptoms of malaria, have probably acquired their infections during the transmission season. In high transmission settings, microscopy can identify a substantial proportion of asymptomatic infections while in low transmission settings molecular methods are needed as most infection are sub-microscopic , 21. InfNational Malaria Control Programs (NMCP) in collaboration with academic institutions should estimate the magnitude of the problem of residual transmission, including information on human and vector behavior, and intervention effectiveness . As the This cohort study was carried out in six sites across The Gambia, in the western, central and eastern part of the country, further subdivided into the north and south banks. This division corresponded roughly to five of the seven administrative regions, namely West Coast (WCR), North Bank (NBR), Lower River (LRR), Central River (CRR) and Upper River (URR) Regions, with the latter further divided in the north (URR-N) and south (URR-S) bank. In each of the study sites, we carried out a cross-sectional survey in November 2012, at the peak of the transmission season , in six In April 2013, following village sensitization meetings, all residents (as confirmed by household heads), aged ≥6 months were recruited after provision of individual informed consent (parents for children) and assent for children ≥12 years old. Individuals with chronic medical conditions were also included. Recruitment lasted three months (May-July 2013) to maximize coverage. Case record forms (CRFs) were pre-tested in March 2013, prior to starting the study, to evaluate the logical flow of the questions and correct interpretation of each question into the local language by the field coordinator and the understanding of the questions by the interviewees P. falciparum infections were detected by diagnostic nested PCR from the collected dry blood spots. Asymptomatic malaria infections were defined as positive nested PCR in individuals without fever (or other acute symptoms), and no history of recent antimalarial treatment [Monthly surveys, in which all residents present in the villages were included, were carried out during the last 2 weeks of each month, between June and December 2013; an additional survey was done in April 2014, during the dry season. At each survey, information on the use of LLIN, sleeping habits (outdoors), malaria symptoms, antimalarial treatment, and travel history was collected. Village health workers, one per village, helped the study nurses by providing information on participants who travelled, or migrated out of the village or had died. Axillary temperature was measured using a digital thermometer. A blood sample was collected by finger prick for haemoglobin measurement, malaria diagnosis by microscopy and molecular analysis on dry blood spots filter paper, . Clinical malaria patients were identified by passive case detection at local health facilities or in villages by study nurses, and were treated with artemether-lumefantrine. Clinical malaria was defined as a history of fever in the previous 24 hours and / or axillary temperature ≥ 37.5°C with a positive Rapid Diagnostic Test (RDT) . Human behaviour was assessed by asking about travel history outside the village and sleeping outdoors at night in the previous 30 days. A household was defined as a group of individuals living in the same building and eating from the same pot. The structure of each house was defined as traditional or modern based on the following characteristics; type of roof , type of wall (mud or cement), open or closed eaves, presence of windows and presence or absence of screens on the windows. The proportion of houses with each of these variables was defined as the number of houses with that specific variable divided by the total number of houses in each village. LLIN use, sleeping outdoors at night, travel outside the village were defined as the number of individuals that reported LLIN use the previous night, sleeping outdoors and travelling any time during the month by the total number of individuals surveyed, respectively. reatment . Sub-patFor diagnostic nested PCR, three 6mm dried filter paper blood spots (DBS) were punched into a 96-well plate. DNA was extracted from the dry blood spots using the automated QIAxtractor robot (Qiagen). Negative and positive (3D7) controls were included to control for cross-contamination and DNA extraction efficiency, respectively. The blood spots were lysed by incubating them in tissue digest buffer at 60°C for 1 hour and digested eluates were applied onto capture plates, washed, and the DNA eluted into 80μl.The extracted DNA (4μl) was used in a nested PCR amplifying the multi-copy Plasmodium ribosomal RNA gene sequences using genus and species specific primers , 29. AllThick blood smears were stained with 2% Giemsa for 30 minutes and examined by two independent microscopists. Discrepancies were settled by a third reader. Parasite density was estimated by counting the number of parasites per 200 WBC, and assuming 8,000 WBC/μL of blood. Blood smears were considered negative if no parasites were found after reading 100 high power fields.An. gambiae sensu lato (s.l) mosquitoes were separated from An. funestus and stored in separate tubes with silica gel. Other anophelines and culicine mosquitoes were counted and discarded. An. gambiae s.l. females captured by HLCs were dissected to extract the ovaries and determine parity [An. gambiae s.l. head and thorax samples were used for the detection of P. falciparum circumsporozoite protein (CSP) by ELISA [Monthly entomological sampling was done from 7pm to 7am by human landing catches (HLCs) and CDC light trap catches (LTC). Indoor and outdoor HLCs were conducted in two houses per village for three consecutive nights (n = 6). Upon completion of the HLCs, LTCs were set up in six houses per village on alternative nights for two nights (n = 12). Each morning, mosquitoes were counted and species identified morphologically. The e parity , 33. An.by ELISA .P. falciparum infection  as determined by nested PCR.The primary outcome was incidence of Secondary outcomes included; incidence and prevalence of clinical malaria, prevalence of asymptomatic malaria infections, gametocytes, and sub-patent infections.Entomological outcomes included; entomological inoculation rate (EIR) and vector parity.® database and analysed using Stata 14 . Recurrent episodes of P. falciparum infection were categorized as 1, 2, and ≥ 3 episodes per individual. Anaemia was categorized as mild, moderate or severe according the WHO age-specific criteria [Data were double entered into a Microsoft Accesscriteria . Binary P. falciparum infection were calculated by dividing the number of new infections  by person-years. Incidence rate ratios (IRRs) compared the incidence of P. falciparum infection and clinical malaria between WCR and the other regions . A Cox proportional hazards model and Kaplan-Meier survival curves were used to estimate the risk of clinical malaria among members within the same household with or without at least a malaria-infected individual at the start of the transmission season (June-July). Cox proportional hazards models with a shared frailty at region level and controlled for changes in variables (e.g. LLIN use) over time were used to predict the factors associated with P. falciparum infection at the start of the transmission season (June-July) and factors associated with sub-patent infection. A Poisson model was used to predict individual’s and household’s risk factors of recurrent malaria infections. Confidence intervals for gametocyte prevalence were calculated by the Wilson Score method with continuity correction. The sporozoite rate was the proportion of P. falciparum CSP positive mosquitoes divided by the total number of mosquitoes caught. The human-biting rate (HBR) was expressed as the number of bites per person per night (b/p/n) determined by dividing the number of mosquitoes collected by the number of volunteers per night. The entomological inoculation rate (EIR) was estimated by multiplying the sporozoite rate by the human-biting rate [Monthly incidence rates of ing rate . Vector Parity was defined as the proportion (p) of parous vectors divided by the total collected by HLC and LTC (p/np+p), by region. Pairwise Binary data between vector parity and incidence of malaria infection were compared using a Pearson chi-squared test and the correlations were based on the Spearman rank based approach.Verbal consent from the study communities was obtained at village meetings prior to field activities. Written informed consent was obtained from all participants ≥ 18 years old and assent from children aged 11–17 years. Parents/guardians provided written consent for children <18 years. All households selected for LTCs and volunteers for HLCs provided additional written informed consent. The study was approved by the Scientific Coordinating Committee of the Medical Research Council Unit The Gambia (SCC 1318), and the Gambia Government/MRC Joint Ethics Committee.In June 2013, a total of 4,194 residents were enrolled from 426 households across the twelve study villages Sleeping outdoors was high in July  but declined significantly in September  and November  .At the start of the transmission season, in June and July, the overall malaria prevalence was 4.94%  and 4.86% , respectively. At this time, the risk of malaria infections was significantly lower in females (Hazard Ratio (HR) = 0.72, 95% CI: 0.54–0.97, p = 0.03) than males, among LLIN owners , than in individuals without LLINs and lower in individuals sleeping under an LLIN ; it was significantly higher among those who had travelled outside the village  and in those who slept outdoors . During the entire transmission season, about a third  of infections were sub-patent, the highest proportion found in June  and July , at the beginning of the transmission season, while the lowest was in October  . Sub-patMalaria prevalence during the dry season (April 2014) was 6.68% (212/3173), and 60.37% (128/212) of these infections were sub-patent. Most infected individuals in April  were negative in December 2013.The overall incidence rate of malaria infections was 1.01 infections/PYAR (95% CI: 0.94–1.17) and that of clinical malaria was 0.38 infections/PYAR (95% CI: 0.36–0.43). Incidence of malaria infections was low in July , decreased significantly in August  and September , p<0.01, and increased steadily in October , November  and December  (p<0.01). Similarly, the incidence of clinical malaria increased significantly from August  to December  with the peak in November  (p<0.01), when compared to July. .Malaria prevalence in July was similar and not statistically different across the regions , with the exception of the URR-S  and URR-N  where it was significantly higher than in the WCR.P. falciparum infection and clinical malaria incidence during the transmission season differed substantially between regions  and NBR  and the lowest in URR-N  and URR-S  . Sub-patNone of the infections identified in June carried gametocytes; however, the proportion of infections with gametocytes increased significantly, from 9.39% (17/181) in July, to 21.69% (113/521 p<0.01) in November  S1 Tabl. During P. falciparum infection; 5.36% (224/4179) had two and 2.46% (103/4179) had three or more episodes. Multiple episodes of malaria infections were significantly more frequent in the URR-N  and URR-S  than in WCR  (p<0.01). Older children (5 to 14.9 years)  had a higher risk of multiple episodes and adults ≥30years a lower risk  than younger children. Similarly, females  and LLIN owners  had a lower risk of multiple episodes of infection  had a single episode of nfection .In households with at least one malaria-infected individual at the beginning of the transmission season, the household risk of clinical malaria for other household members was significantly higher  than in the rest of the population, regardless of their infection status  An. gambiae s.l. was the main vector in all regions, with the exception of CRR where An. funestus was the most predominant one . Indoor biting, after weighing for sleeping indoors or outdoors, was higher than outdoors biting, in all regions. Indoor biting was significantly higher in the WCR than in other regions (p<0.01) except in the CRR (p = 0.74)  by HLC.  = 0.74) .Indoor biting was significantly lower in July than in each of the following months, with more than 90% of indoor biting between August and November .A substantial proportion of biting occurred between 4-6am in all regions Similarly for each vector species, substantial proportion of indoor and outdoor biting occurred between 4am-6am An. gambiae s.l. An. coluzzii was the predominant species in western (WCR and NBR) and eastern (URR-S and URR-N) Gambia while An. gambiae s.s. was most common in the other two regions, LRR and CRR, in the centre of The Gambia. An arabiensis was mainly found in the central and eastern regions (Speciation by PCR was successful for 65.43% (1963/3000) of the  regions .The species composition varied substantially during the transmission season 2 0.7, p<0.01) but not with that of clinical malaria  (The unadjusted vector parity was correlated (Spearman) with the incidence of malaria infection (rp = 0.3) An. gambiae s.s. 6%; An. arabiensis 9%; An. coluzzii 67%) and deltametrin  was more common in eastern Gambia [The prevalence and incidence of malaria infection and clinical disease during the 2013 transmission season were significantly different across the six study sites, despite similar high coverage of control interventions. Mass distribution of LLINs was carried out in 2009, 2011 and 2014, and annual indoor residual spraying (IRS) with DDT since 2009; the last cycle was implemented in August 2013 in all rural regions . The mosn Gambia . Howevern Gambia .An. arabiensis and An. coluzzii represented together about 80% of the vector population in URR, where the incidence of infection and clinical malaria was the highest. These two species are well adapted to the semi-arid conditions found in the eastern region and are highly efficient at transmitting infection during the short transmission season. An. coluzzii is anthropophilic, endophilic, endophagic, and late-night biting [An. arabiensis is more zoophilic, exophily, exophaghic, and early-night biting [An. gambiae s.s in the URR may be the result of out-competition from other species. Entomological surveys in Gambia have shown heterogeneity in An. gambiae s.l species composition [Differences in species composition between and within the different regions may further explain the observed residual transmission and high endemicity in the eastern region. t biting  while Ant biting , though position , 42 withposition . Therefoposition . The higposition  a behaviposition , 45. SucMalaria prevalence in the dry season was similar to that observed at the beginning of the 2013 transmission season. However, considering that most individuals infected in April 2014 were malaria negative in December 2013, their infection was probably acquired at the very end of the transmission season, when incidence of infection was the highest; thus most infections identified in December were either cleared over the next 2–3 months or under the detection threshold in April . IndividThough malaria prevalence was strikingly similar in June 2013 (5.0%) and April 2014 (6.7%), gametocyte carriage at these two time points was extremely different, 0% and 14%, respectively. It is unclear whether individuals infected in April 2014, who possibly restart malaria transmission at the onset of rains, would gradually clear gametocytes. In the 2003 dry season, in Farafenni, about half of the 37 infections detected carried gametocytes; interestingly the proportion of infections with gametocytes did not vary by season though infectivity to mosquitoes varied . SeasonaThe risk of malaria infection at the start of the transmission season was higher among individuals who had travelled outside the study villages. This highlights the importance of human mobility in maintaining transmission, especially in settings aiming at achieving pre-elimination . Women hThe risk of clinical malaria was higher in households with at least one malaria-infected individual at the beginning of the transmission season. Though this association, after adjusting for several confounding factors, remained significant only in two regions, it probably indicates the higher risk of exposure to malaria in these households, possibly because of their location in relation to breeding sites or their structure. Such clustering of malaria cases has been previously reported in a pooled analysis, showing that household members of index malaria cases were overall five-times more likely to have malaria than members of other households . In KenyOlder children were at higher risk of multiple episodes of malaria infections, an observation already made in settings with low malaria transmission. , 54 StudThe EIR is considered a standard metric of malaria transmission.  However,The follow up of the study population was intense  and challenging. Some groups were more difficult to follow up than others, for example adult males were more often traveling than females and children, and this could have affected some risk estimates. However, the overall loss to follow up was relatively low (11.0%) given the study design and unlikely to significantly affect the study outcomes.In conclusion, there is still significant residual malaria transmission across The Gambia, particularly in the eastern region. This is maintained by a low-density vector population with high survival, despite the high coverage of conventional vector control interventions. Even in sites where transmission is significantly lower than in eastern Gambia, a non-negligible proportion of individuals maintain malaria infections, from the previous transmission season to the next one. Such individuals should be identified and targeted with additional interventions aiming at reducing the human reservoir of infection. Additionally, new interventions able to target vectors escaping LLIN and IRS are needed.S1 Table(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Malaria remains the most important cause of morbidity and mortality in Tanzania. However, its prevalence varies from area to area depending on various ecological, socio-economic and health system factors. This study was carried out to determine malaria prevalence and associated factors among rice farming communities in the Kilangali village of Kilosa District in Central Tanzania.A cross-sectional study was conducted in May 2015, involving randomly selected persons living in the six sub-villages of the Kilangali village, namely Mlegeni, Kisiwani, Makuruwili, Kwamtunga, Upogoroni and Chamwino. A finger prick blood sample was obtained for diagnosis of malaria infection using Giemsa-stained thick smears and a rapid malaria diagnostic test. Study participants were also screened for haemoglobin levels and a total of 570 children aged ≤ 12 years of age were examined for spleen enlargement using the palpation method.Plasmodium falciparum accounted for the majority (89%) of the malaria infections. The overall geometrical mean parasite density was 20.5 parasites/μL (95% CI: 14.6–28.8). Malaria prevalence and parasitaemia was highest among individuals living in the Mlegeni (23.9%) and Makuruwili (24.4%) sub-villages. Among the children examined for splenomegaly, 2.98% (17/570) had it. The overall prevalence of anaemia was 34.6%. Malaria infection was associated with the age groups of 1–10 years  and 11–20 years ; and mild anaemia  and moderate anaemia .A total of 1154 persons were examined for malaria infection with mean age of 21.9 ± 19.69 years. The overall malaria prevalence was 14.2% and 17.5% based on microscopic examination and rapid diagnostic test, respectively. Malaria was found at the study setting and its prevalence varied according to the demographic characteristics of the study participants and between sub-villages that are closely located.The online version of this article (doi:10.1186/s40249-017-0315-1) contains supplementary material, which is available to authorized users. Please see Additional file Intensive use of malaria intervention measures has resulted in the decline of malaria transmission in some parts of the world, including in Sub-Saharan Africa , 2. ReceThere are variations in malaria transmission between regions of Tanzania , 10, witIn Tanzania, malaria is mostly a disease affecting rural populations characterised by a poor health care system and where agriculture forms the backbone of the household economy . In thesIn preparation for a large study, focusing on integrating the application of biolarvicides and fertiliser in rice fields to control malaria and increase rice yields in Tanzania, it was deemed necessary to generate baseline information on malaria prevalence in order to assess the effect of the intervention on malaria before and after the application of biolarvicides. Thus, the present study was conducted to determine the malaria prevalence and associated factors in the Kilangali village in Kilosa district of Central Tanzania.This study was carried out in the Kilosa district of Central Tanzania  see Fig.  in May 2This was an analytical cross-sectional survey conducted in the Kilangali village, Kilosa district, and included individuals of all age groups selected randomly from 253 out of 750 households located in the six sub-villages. Selected participants were included in the study if they had been living in the sub-villages for more than 2 years (considered as permanent inhabitants). Considering, on average, that a household has four residents , a samplDemographic information of the study participants, which included age, sex, place of residence, history of malaria treatment in the past four weeks and whether or not the participant slept under a mosquito net the night before the survey, was collected.School children were examined for malaria infection in their school environment and other members of the community were examined at a specified point in the sub-village. A finger prick blood sample was obtained from all study participants, following aseptic procedures. Both malaria rapid diagnostic tests (mRDTs) and microscopy were employed for diagnosis. Malaria diagnosis was done using a mRDT  and the procedures were performed according to the manufacturer’s instructions. From the same blood sample, thick and thin blood smears was prepared and stained with 3% Giemsa staining solution and examined under a light microscope with oil immersion for the presence of malaria parasite stages and quantification of malaria parasitaemia. A Giemsa-stained thin smear was used for the identification of malaria parasite species. Two laboratory technicians examined the slides independently and for quality assurance, 10% of the randomly selected positive and negative slides were re-examined by a third laboratory technician. Slides were considered negative for malaria infection if no parasites were seen in at least 200 oil immersion fields on the thick film. Malaria parasite density was determined according to the number of parasites per 200 white blood cells (WBCs), assuming a total WBC count of 8000/μL .www.EKF-diagnostic.com). Anaemia was defined as Hb < 11 g/dL for all study participants [A sub-sample of the finger prick blood samples was used to determine the haemoglobin (Hb) level. Haemoglobin levels were measured using the Hemo-Control® system  or Fisher’s exact test where appropriate. Comparison of means or geometrical mean malaria parasite densities was done using t-tests for two variables and analysis of variance (ANOVA) for comparison of more than two variables. For logistic regression, factors with P-values of 0.2 were considered for the multivariable analysis. Factors such as sex, age in years, presence of anaemia and use of mosquito nets were considered for the multivariable analysis. Because of the interaction between splenomegaly and age, a splenomegaly variable was not considered for the multivariable analysis. The World Health Organization cut-off for Hb was used to classify anaemia [Data were double entered into Epi Info™ version 6  and then transferred to Stata® version 13 . Frequencies and percentages were used to describe the data. Continuous variables were described using median, interquartile range (IQR) and mean ± standard deviations. Categorical variables were described using the chi-square test . Study participants who reported not using mosquito nets had the highest prevalence of malaria parasites and had higher geometric mean parasite densities than those who reported using mosquito nets .A total of 86.7% of the study participants reported to own and use mosquito nets. There was a difference in terms of sex in relation to the reported use of mosquito nets, with female participants having a higher rate (55.9%) of using mosquito nets than males (44.1%) , P. malariae accounted for 3.7% of the infections and 7.3% were mixed infections of P. falciparum and P. malariae. The overall prevalence of P. falciparum malaria infection was 14.2% based on microscopic examination and the geometric mean parasite density was 20.5 parasites/μL (95% CI: 14.6–28.8), with a marginal difference between the age groups  and significant differences between the sub-villages . Based on the mRDT results, the prevalence of P. falciparum infection was 17.5%. In general, 12.8% (n = 21/164) of the study participants infected with malaria parasites had gametocytes.P = 0.001). The age groups of 11–20 years (26.8%) and 1–10 years (22.5%) had the highest prevalence of malaria (see Table P = 0.011), sub-village of residence (P = 0.031) and reported use of mosquito nets (P = 0.004).There was a significant difference in the prevalence of malaria between male and female participants, with males having a higher prevalence P = 0.00. The ageP = 0.001). Study participants from Kisiwani (47.9%) and Makuruwili (42.4%) with anaemia had the highest prevalence of malaria (P = 0.001). Of the study participants with anaemia, 44.9% had malaria.Only a total of 1083 out of 1154 study participants were screened for haemoglobin level because Hemo-cuvette for estimation of Hb levels using the Hemo-control system were only available for that number of study participants. About one-third (34.6%) of the study participants were anaemic (Hb < 11 g/dL). Of the anaemic participants, 4% (15/375), 49% (184/375) and 47% (176/375) were classified as having severe, moderate and mild anaemia, respectively. In relation to malaria infection, 3.5, 47.1 and 49.4% of the study participants with severe, moderate and mild anaemia were infected with malaria. The age groups of 1–10 years (48.6%) and 11–20 years (24.7%) had the highest prevalence of anaemia . All study participants who had splenomegaly were in the age groups of 1–10 years (2.1%) or 11–20 years (5.1%). Of the study participants with splenomegaly, 8.9% had malaria.OR = 4.41, 95% confidence interval, CI: 1.96–9.93, P = 0.001) or 11–20 years , and having mild  or moderate anaemia  remained independently associated with malaria infection.In the bivariate analysis, malaria infection was mainly associated with being male, in the age group of 1–10 years or 11–20 years, having splenomegaly, and having moderate or mild anaemia (see Table P. falciparum accounts for the majority of the cases. In Tanzania, P. falciparum accounts for more than 95% of the malaria cases reported in health facilities [P. falciparum parasites have been repeatedly observed to be the leading cause of symptomatic and asymptomatic malaria infections and disease [Anopheles gambiae, in Africa [The findings of the present study indicate that there is malaria infection in the Kilangali village and that cilities . Similar disease , 21, 22.P. falciparum malaria observed varied significantly according to age and sex, with the youngest age groups having a higher prevalence of malaria. Recent studies in the same district [The prevalence of district , 22 and district  have obsdistrict . Howeverdistrict , 24. Thedistrict .In this study, males had a higher prevalence of malaria infection than females. This is mostly likely to be a reflection of the variation in behaviour related to exposure . Our ownP. falciparum malaria was higher among inhabitants of Makuruwili and Mlegeni, which are located close to a large commercial rice seed irrigation scheme. Other authors have demonstrated higher malaria prevalence in inhabitants living in households located close to traditional flooded rice irrigation schemes [As expected, our findings have demonstrated a significant difference in malaria prevalence and parasitaemia between the sub-villages. Prevalence of  schemes , 22. Sig schemes  and in pAnaemia was also prevalent among the study participants and the youngest age group had the highest prevalence. In addition, the prevalence of anaemia varied significantly by village of residence, with study participants from the sub-villages of Kisiwani and Makuruwili having the highest prevalence of anaemia. Previous studies have recorded high prevalence of anaemia among school-age children from rice farming communities in the Kilosa , 22 and As shown in previous studies , 32, 35,The present study was not conducted without limitations. The use of expert microscopy and rapid diagnostic tests may have missed light infection when compared to using sensitive molecular methods such as polymerase chain reactions . In addiOur results indicate that there is malaria infection in the study setting and that the prevalence varies according to age group, sex, sub-village of residence and reported use of mosquito nets. Further studies are needed to understand the observed variation in malaria prevalence and contribution of household and socio-economic factors.


Plasmodium species that infect humans, adverse effects of P. falciparum and P. vivax have been extensively studied and reported with respect to poor outcomes particularly in first time mothers and in pregnant women living in areas with unstable malaria transmission. Although, other non-falciparum malaria infections during pregnancy have sometimes been reported, little is known about the dynamics of these infections during pregnancy.Among the Plasmodium spp. Risk factors associated with Plasmodium spp. infection during pregnancy were assessed as well as the relationships with pregnancy outcomes.Using a quantitative PCR approach, blood samples collected from Beninese pregnant women during the first antenatal visit (ANV) and at delivery including placental blood were screened for P. falciparum was the most prevalent Plasmodium species detected during pregnancy, irrespective either of parity, of age or of season during which the infection occurred. Although no P. vivax infections were detected in this cohort, P. malariae (9.2%) and P. ovale (5.8%) infections were observed in samples collected during the first ANV. These non-falciparum infections were also detected in maternal peripheral blood  at delivery. Importantly, higher prevalence of P. malariae (5.5%) was observed in placental than peripheral blood while that of P. ovale was similar . Among the non-falciparum infected pregnant women with paired peripheral and placental samples, P. malariae infections in the placental blood was significantly higher than in the peripheral blood, suggesting a possible affinity of P. malariae for the placenta. However, no assoctiation of non-falciparum infections and the pregnancy outcomes was observedPlasmodium spp. infection during pregnancy, indicating placental infection by non-falciparum Plasmodium and the lack of association of these infections with adverse pregnancy outcomes.Overall this study provided insights into the molecular epidemiology of  P. falciparum and P. vivax infections during pregnancy have been extensively studied. Meanwhile, the dynamics of other non-falciparum malaria infections during pregnancy is not well understood. We investigated the prevalence of Plasmodium spp. in samples collected from Beninese women early in pregnancy and at delivery using a quantitative PCR approach. Factors associated with Plasmodium spp. infection during pregnancy were assessed. P. falciparum was the most prevalent Plasmodium species detected. P. malariae and P. ovale infections were also detected early in pregnancy, and in the maternal peripheral and placental blood at delivery. Noteworthily, the high prevalence of P. malariae in the placenta of pregnant women with paired peripheral and placental samples, suggests a placental affinity of P. malariae during pregnancy. However, association of non-falciparum infections with pregnancy outcomes was not observed. This study provided insights into the molecular epidemiology of Plasmodium spp. infection during pregnancy, and highlighted the underestimated prevalence of non-falciparum infections in pregnancy-related malaria. Plasmodium falciparum is responsible for most cases of malaria during pregnancy and is linked with poor outcomes both among mothers and their babies [P. falciparum infected erythrocytes to sequester in the placenta, inducing a placental inflammation that may lead to foetal growth alteration, stillbirth, and low birth weight (LBW) of the babies [r babies ,2. Pregne babies –5. Futhee babies ,7.Plasmodium parasites that infect humans , P. vivax has been reported in Asia and Latin America to have consequences during pregnancy, such as maternal anemia and LBW deliveries [P. vivax sequestration in the placenta [P. malariae and P. ovale in pregnant women [P. falciparum does. Thus, geographical distribution of non-falciparum Plasmodia in West Africa, where P. falciparum is the most prevalent malaria species, and their involvement in the pathogenesis of pregnancy-associated malaria need to be documented. A PCR-based approach was used to better detect the non-falciparum malaria parasites due to their low parasite densities and the difficulty to accurately differentiate the species by morphological analysis using microscopy [falciparum malaria parasites in the placental blood [Among the other liveries –12. Howeplacenta . The detnt women –16 has rcroscopy . Only feal blood ,18,19.P. falciparum [Plasmodium spp. infection early in pregnancy , and at delivery in maternal peripheral and placental blood samples collected from a cohort of Beninese pregnant women across different malaria transmission seasons.Current efforts to prevent and control malaria during pregnancy using drug-based preventive treatment or vaccine strategies essentially target lciparum . In thisThe Strategies To Prevent Pregnancy Associated Malaria (STOPPAM) project was approved by the Comité Consultatif de Déontologie et d’Ethique of the Research Institute for Development (France) and the ethical committee of the Faculty of Health Science . All procedures complied with European and French national regulations. Written informed consent was given by participants who were all adults.Plasmodium spp. The blood smears were Giemsa-stained in phosphate-buffered saline (PBS) and examined by two independent microscopists. A third read was performed in case of discrepancies between the first two reads. Two hundred microliters of blood pellet were stored at -20°C for DNA extraction.Samples used in this study were collected from pregnant women during the STOPPAM study conducted from 2008 to 2010 in Southern Benin. Details of the project have been reported elsewhere . BrieflyBased on the available data of the parameters to be considered in this study, 975 peripheral blood samples collected at enrolment, and 667 peripheral and 562 placental blood samples collected at delivery were analysed. No placenta histology data was available for this study. Genomic DNA (gDNA) was extracted from the frozen whole blood using the DNAeasy Blood & Tissue kit (Qiagen), as recommended by the manufacturer.Plasmodium spp. were detected in the whole blood DNA samples as described [Plasmodium-specific primers and probes previously published [GAPDH gene GAPDH_Fw: CCTCCCGCTTCGCTCTCT, GAPDH_Rev: GCTGGCGACGCAAAAGA) and GapdhProbe: VIC-CCTCCTGTTCGACAGTCAGCCGC–MGBNFQ). GAPDH was used as an internal control gene to ensure that gDNA was successfully extracted. Plasmodium load was quantified by extrapolation of cycle thresholds (Ct) from a 6 fold standard curve of Plasmodium ring-infected erythrocytes. Samples without amplification (no Ct detected) were considered negative, and a density of 2 parasites/μl was assigned if amplification was observed out of the lower range of the standard curve (5 parasites/μl). A negative control with no DNA template was run in all reactions. This study benefited from a quality check program that was established to ensure correct performance of qPCR techniques between several laboratories including ours [Species of escribed . Brieflyublished , and a ding ours .Plasmodium species  were detected in a multiplex PCR reaction using species-specific forward and reverse primers, as well as specific probes, as described by Taylor et al. [P. ovale curtisi and P. ovale wallikeri were used to amplify both variants of P. ovale [P. vivax detection, samples were pooled into group of 10 samples and screened as single PCR reaction. Amplification was performed using P. vivax specific primers and probe under the same conditions described above with few modifications. A total of 45 cycles was performed, and samples from any pool with amplifications have been individually screened for P. vivax detection.r et al. . SpecifiP. ovale . For P. Plasmodium spp. infection at different time-points of collection were determined according to the age and the parity of the pregnant women. The influence of the season and intensity of malaria transmission were explored in relation to Plasmodium spp. infections among pregnant women. Months in the year were coded from 1 to 12 respectively for January to December. Dry seasons were defined as December-March and August  while wet seasons were defined as April-July (4–7) and Sep-Nov (9–11), as previously described [P. ovale and P. malariae infections were combined and analyzed as non-falciparum infections. Infections involving P. falciparum and non-falciparum parasites together were grouped as mixed infections. Furthermore, a multi-variable logistic regression model was used to assess the risk factors for P. falciparum, non-falciparum and mixed infections at different time points by controlling for potential confounders and using those without malaria infection as the reference group. In addition, an exploratory analysis was conducted at selected time-points to determine whether there was any association between Plasmodium spp. infections and primary pregnancy outcomes , and active PM. For this analysis, groups of women presenting each outcome were considered, and the distribution of P. falciparum and non-falciparum in the detected infections early in pregnancy and at delivery was investigated in comparison to women with no malaria infection from the corresponding outcome group. Active microscopic PM was defined as confirmed Plasmodium spp. infection in placental blood by microscopy, and was found to be exclusively P. falciparum active PM. All statistical tests were conducted at 0.05 level of significance.Statistical analysis was conducted using STATA software version 13. Prevalence of escribed . P. ovalPlasmodium spp. as well as the parasite load of the infections determined by microscopy, are reported in Parasites density generated from qPCR amplification of P. falciparum was the most prevalent Plasmodium species found in infected women. P. falciparum infection was detected in 39.3% of samples analysed either as a mono-species infection (29.6%) or involved in mixed infection (9.7%) with other Plasmodium species . When the analysis was performed considering the non-falciparum Plasmodium parasites as mono-species infection, a reverse trend was observed. Indeed, the prevalence of P. malariae mono-infection (1.4%) was significantly lower as compared to P. ovale mono-infection (2.3%) . Mixed infections involving P. falciparum and non-falciparum were detected in 98 (10.1%) samples as reported in P. vivax was not detected in samples analysed in this work.Among samples collected early in pregnancy at the inclusion time-point,  species . InfectiPlasmodium spp. infections at enrolment was analysed according to the parity of the women and their age  and age , resulting in higher prevalence of P. falciparum infection in primigravid and younger women. No relation was observed when non-falciparum infections at enrolment were compared with parity and age of the women.The prevalence of heir age . The prePlasmodium spp. infection at delivery was assessed in the peripheral and placental blood samples. P. falciparum was, by far, the most prevalent Plasmodium species detected in maternal peripheral (34.6%) and placental blood (32%) samples  was significantly higher than those involving P. ovale (1.8%) . Mixed infections comprising P. falciparum/P. malariae; P. falciparum/P. ovale; P. falciparum/P. malariae/P. ovale and P. malariae/P. ovale were respectively detected in 12 (2.1%); 4 (0.7%); 2 (0.4%) and 2 (0.4%) placental samples. Most importantly, mono-infections with P. malariae were detected in the placenta of 15 women (2.7%), at a higher prevalence compared to that of P. ovale (0.4%) . However, this difference was not observed in the maternal peripheral samples . As at enrolment, no P. vivax infection was detected in the samples collected at delivery. samples . PlasmodP. falciparum nor non-falciparum infections were associated with parity or age of the women. However, younger mothers were the most exposed to mixed infections (p < 0.001), while no difference was observed in the prevalence of P. falciparum and non-falciparum infections over different age groups and the parity of the women , suggesting that younger pregnant women are more susceptible to develop a placental P. falciparum-malaria although being co-infected with other Plasmodium species. It is worth noting that although not statistically significant, primigravid women had higher prevalence of P. falciparum than multigravid women.P. falciparum and non-falciparum infections was analysed according to seasons corresponding to the blood sampling. The monthly prevalences of P. falciparum and non-falciparum infections at enrolment and at delivery are shown in P. falciparum infections  was significantly higher than non-falciparum infections  . This differential monthly prevalence of P. falciparum and non-falciparum infections was maintained at delivery with a high prevalence of P. falciparum infections in peripheral and placental blood samples . However, over three successive months (January to March 2010), similar prevalence values of P. falciparum and non-falciparum placental infections were observed. Of note, Plasmodium spp. infection early in pregnancy clearly appeared to be high regardless of season and irrespective to the Plasmodium species. In these samples collected at enrolment, no distinct pattern of overlapping peaks of Plasmodium spp. prevalence and the rainy seasons was observed, although a significant increase of mixed infections (p = 0.045) was detected between September and November  women had a significantly lower risk of P. falciparum infection early in pregnancy . Parity, gestational age and age of the women did not significantly influence non-falciparum infections at enrolment, probably due to the limited size of this group , as compared to uninfected women at enrolment , and a similar trend was found for P. falciparum infected mothers at enrolment and at delivery.The relationships between d Tables  and 8. Tnrolment . Notewornrolment . No relafalciparum infection at delivery, 38  and 18  paired peripheral and placental samples were analysed. P. malariae infections rate in the placental blood was significantly higher than in the peripheral samples   (Among pregnant women with non- 0.0001) . A similnsidered . In a di samples . However 0.0001) .falciparum malaria parasites is under re-evaluation due to continued development of more sensitive PCR-based diagnostic tools. Traditionally cited as uncommon causes of malaria in many African areas, P. malariae, P. ovale and P. vivax infections have been reported at increasing prevalences [falciparum infections include chronicity, low parasite density, asymptomatic and multiple species infection [falciparum infections among different populations remains undocumented in many West African countries, including Benin. We described the prevalence of different species of Plasmodium parasites that infect Beninese women during pregnancy, and confirm the presence of infections with P. ovale and P. malariae parasites among West African pregnant women, as previously reported [P. ovale and P. malariae parasites were detected either as mono-infections, as dual infections or as mixed infections with P. falciparum in 3.7%, 0.4% and 9.3%, respectively, of pregnant women at first ANV, leading to a prevalence of non-falciparum malaria infection of 13.4%. This prevalence is higher than in other West African countries  [falciparum infection might be explained by differences in study design and different epidemiological features, as well as by the highly sensitive PCR approach used [The geographical distribution of non-valences –29. Commnfection . In Westnfection ,32, adulnfection , and prenfection –16. Howe, 0.72%) . This hiach used ,16.P. vivax infection was detected in pregnant women, although circulating P. vivax among non-pregnant Beninese was reported [P. vivax selected a group based on sero-reactivity to rPvMSP1 and rPvCSP1 antigens [P. vivax in a non-biased population.No reported . The autantigens , thus itP. falciparum was the most prevalent Plasmodium spp. supporting the general consensus that P. falciparum is responsible for most cases of pregnancy-related malaria. Indeed, P. falciparum infections either at enrolment or at delivery were related to active microscopically detectable PM, as reported [P. falciparum infection decreased with increasing age or parity, confirming the age- and parity-related susceptibility to malaria during pregnancy. Strikingly, this parity effect disappeared at delivery, suggesting a similar risk of P. falciparum infection for all pregnant women in the last weeks of pregnancy. This lack of parity-related susceptibility at delivery might be the effect of IPTp, which clears subsequent infections, but fails to protect against re-infections after the second dose of SP [P. falciparum placental infection which may persist from early pregnancy, since no age-related difference was observed in the prevalence of infection in maternal peripheral blood. This finding highlights the influence of IPTp, by reducing the prevalence of infection until delivery. On the other hand, this observation may also suggest that most Plasmodium spp. infections at delivery derived from new infections in IPTp-implemented areas.Both at enrolment and delivery, reported ,35. At ese of SP ,37. In fse of SP . An incrse of SP  has beense of SP ,39. Our falciparum malaria infection both early in pregnancy and at delivery, both in maternal peripheral and placental blood. In previous reports, P. malariae and P. ovale have been detected in placentas and placental blood samples [P. malariae and P. ovale in pregnant women at delivery, either as mixed or as mono-infections. The higher prevalence of P. malariae in the placental compartment compared with peripheral blood suggests a potential affinity of P. malariae for the placenta. This hypothesis requires further investigations to characterize P. malariae parasites, and to demonstrate any association with placental pathology.A major feature of our data is the detailed description of non- samples ,40 from Plasmodium spp. infection, the prevalence of mixed-Plasmodium infections was lower during the first rainy season. Similarly, a lower prevalence of non-falciparum, mainly P. malariae, was detected during both rainy seasons as compared to the dry season. At delivery, prevalence of non-falciparum placental blood infection was lower in the first rainy season. One limitation of this study is the lack of contemporary rainfall and entomological data at sample collection. Such information would allow to better define rainy and dry seasons, and to better correlate parasite prevalence and malaria transmission. This study showed that epidemiology of malaria early in pregnancy is not strictly dictated by transmission fluctuations, contrary to non-pregnant populations [Plasmodial infection during pregnancy occurs throughout the year in our study area where malaria transmission is continuous . Althougulations . Some inulations ,43.P. falciparum in PM through the placental sequestration of P. falciparum-infected erythrocytes [falciparum and P. falciparum, but not mixed infections early in pregnancy were associated with placental blood infection at delivery. Women infected with non-falciparum at enrolment may be more susceptible to P. falciparum later in pregnancy, leading to PM at delivery. However, longitudinal studies are needed to investigate such putative role of early non-falciparum infection in promoting P. falciparum PM. Mixed infections at delivery displayed distinct relationships with gestational age, enrolment season, and age of the women. Although size differences of the groups may partly account for these observations, the contribution of non-falciparum parasites to the pathology and modulation of the immune response during co-infection with P. falciparum needs to be addressed.Many studies have demonstrated the role of hrocytes –46. HereS1 Checklist(DOCX)Click here for additional data file.S1 TableOR = Odd ratio; *Crude ORs for parity, gestational age, season of enrolment are adjusted for all other Covariates (DOCX)Click here for additional data file.S2 TableOR = Odd ratio; *Crude ORs for parity, gestational age, season of delivery are adjusted for all other Covariates .(DOCX)Click here for additional data file.S3 Table*P value.(DOCX)Click here for additional data file.S4 TableP. falciparum, non-falciparum and mixed infections groups were compared with no malaria infection.* Proportion of women who developed the corresponding outcome from each type of infection group is presented. **(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 Table(DOCX)Click here for additional data file.S8 Table(DOCX)Click here for additional data file.S9 Table(DOCX)Click here for additional data file.


P value <0.05 considered significant. Malaria prevalence values of 20.1%, 43.1%, and 29.7% were obtained for clinical diagnosis, microscopy, and RDTs, respectively (P < 0.05). Values of 47.2%, 95.9%, and 77.8% were obtained for sensitivity, specificity, and diagnostic accuracy, respectively, in clinical diagnosis, while RDTs had sensitivity, specificity, and diagnostic accuracy values of 73.7%, 97.3%, and 88.3%, respectively, when compared to microscopy (P < 0.05). Clinical diagnosed malaria cases should be confirmed with a parasite-based laboratory diagnosis and more qualitative research is needed to explore why clinicians still use clinical diagnosis despite reported cases of its ineffectiveness.The World Health Organization's policy on laboratory test of all suspected malaria cases before treatment has not yielded significant effects in several rural areas of Sub-Saharan Africa due to inadequate diagnostic infrastructure, leading to high morbidity and mortality rates. A cross-sectional randomized study was conducted to evaluate the validity of clinical malaria diagnosis through comparison with microscopy and rapid diagnostic test kits (RDTs) using 1000 consenting outpatients of a tertiary hospital in Nigeria. Physicians conducted clinical diagnosis, and blood samples were collected through venous procedure and analyzed for malaria parasites using Giemsa microscopy and RDT kits. Microscopy was considered the diagnostic “gold standard” and all data obtained were statistically analyzed using Chi-square test with a  Malaria infection causes high levels of morbidity and mortality in Sub-Saharan Africa especially Nigeria . The WorThe study was conducted in Port Harcourt, Rivers State. Port Harcourt is the capital of Rivers State, it lies along the Bonny River in the Niger, Delta region of Nigeria, temperature throughout the year is relatively constant (25°C–28°C), relative humidity fluctuates between 90 and 100%, and it is geographically located at latitude 4.75°N and longitude 7.00°E [μL of blood. A minimum of 200 fields were examined before declaring slides negative for malaria parasite. The RDT kit used was CareStart™ malaria HRP2/pLDH Pf Test Kit  and the usage was according to the manufacturer's instructions. For quality assurance when conducting microscopy, two trained microscopists viewed each blood film before declaring the slide positive or negative but when conflicting results arose, a third senior microscopist was used. The RDT kits were stored at the manufacturer's recommended temperature (<40°C) and quality control and the validity of each kit used was certified by several laboratory scientists. Data accuracy was ascertained by double entry of all data obtained.Clinical diagnosis was conducted by physicians. Blood samples were collected through venous procedure by trained laboratory scientists and stored in Ethylene Diamine Tetra Acetate (EDTA) tubes to prevent coagulation. From the collected blood samples, blood films (thick and thin) were prepared, stained (using Giemsa stain), and examined microscopically using established laboratory procedures . Thick bP value less than 0.05 was considered significant.The data obtained for clinical diagnosis were compared with those for RDT using microscopy as the gold standard to evaluate sensitivity, specificity, diagnostic accuracy, positive predictive value, and negative predictive value using true positives, true negatives, false positives, and false negatives. Data generated were analyzed using Chi-square test and a Plasmodium falciparum was the only species of malaria parasite identified with confirmation from polymerase chain reaction technique (PCR). Prevalence values of 20.1%, 43.1%, and 29.7% obtained for clinical diagnosis, microscopy, and RDTs, respectively (P < 0.05) (P < 0.05) ( < 0.05) . Using m < 0.05) . Plasmodium falciparum. This observation agreed with reports from some similar studies which identified only P. falciparum as the only malaria parasite present [ P. falciparum is the most prevalent malaria parasite in most regions in Sub-Saharan Africa [ Anopheles species thrive well in areas with warm temperature (25°C–28°C) and high relative humidity (90–100%) as well as lack of use of mosquito nets and blocked drainage facilities . The sensitivity, specificity, and diagnostic accuracy recorded for clinical diagnosis was lower than those of RDT using microscopy as the diagnostic standard. There is need to review the use of clinical diagnosis due to its significant underdiagnosis recorded in this study, although some physicians in most malaria endemic regions claim that prompt treatment of suspected malaria cases  reduces the progression of mild malaria to severe malaria. The continual usage of clinical diagnosis alone could have detrimental effects like drug abuse and malaria parasite resistance to antimalarials. RDTs were created to address some shortcomings of microscopy, and some previous similar studies reported low sensitivity (below 100 parasites/μL) and diagnostic accuracy for RDTs [The only malaria parasite observed in this study was present , 13. Accn Africa . The oven Africa , 40.8% in Africa , and 40.n Africa  but lowen Africa , 85.7% in Africa , and 71.n Africa . The stun Africa  and 14.7n Africa . The prefor RDTs , 24 but Clinical diagnosis is not a reliable malaria diagnostic technique especially in Sub-Saharan Africa due to inadequate local epidemiological data on malaria and the presence of other febrile ailments which possess similar signs or symptoms with malaria. Therefore, all suspected malaria cases  should be confirmed with a laboratory test.


We previously demonstrated that endothelial microparticles (EMPs) are increased in mitral valve diseases and impair valvular endothelial cell function. Perioperative systemic inflammation is an important risk factor and complication of cardiac surgery. In this study, we investigate whether EMPs increase in congenital heart diseases to promote inflammation and endothelial dysfunction.The level of plasma EMPs in 20 patients with atrial septal defect (ASD), 23 patients with ventricular septal defect (VSD), and 30 healthy subjects were analyzed by flow cytometry. EMPs generated from human umbilical vascular endothelial cells (HUVECs) were injected into C57BL6 mice, or cultured with HUVECs without or with siRNAs targeting P38 MAPK. The expression and/or phosphorylation of endothelial nitric oxide synthase (eNOS), P38 MAPK, and caveolin-1 in mouse heart and/or in cultured HUVECs were determined. We evaluated generation of nitric oxide (NO) in mouse hearts, and levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cultured HUVECs and in mice.EMPs were significantly elevated in patients with ASD and VSD, especially in those with pulmonary hypertension when compared with controls. EMPs increased caveolin-1 expression and P38 MAPK phosphorylation and decreased eNOS phosphorylation and NO production in mouse hearts. EMPs stimulated P38 MAPK expression, TNF-α and IL-6 production, which were all inhibited by siRNAs targeting P38 MAPK in cultured HUVECs.EMPs were increased in adult patients with congenital heart diseases and may contribute to increased inflammation leading to endothelial dysfunction via P38 MAPK-dependent pathways. This novel data provides a potential therapeutic target to address important complications of surgery of congenial heart disease.The online version of this article (doi:10.1186/s12967-016-1087-2) contains supplementary material, which is available to authorized users. Recently, we demonstrated that circulating microparticles in patients with valvular heart disease impaired endothelium dependent vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase (eNOS) . MicropaPerioperative systemic inflammation is a major risk factor and complication of cardiac surgery. Cardiac surgery with cardiopulmonary bypass (CPB) results in systemic inflammatory responses that contribute to instability of circulation post-operatively , 10. PrePlease refer to the Additional file 20 patients diagnosed with ASD, 23 patients with VSD, and 30 age-matched healthy volunteer control subjects were recruited at The First Affiliated Hospital of Sun Yat-sen University. Patients with diseases which may increase EMPs level, including coronary heart disease, hypertension, infectious disease, severe trauma, antibiotic therapy, lupus anticoagulant, multiple sclerosis, renal failure, rheumatic diseases in acute stage, and valvular heart disease were explicitly excluded. Healthy volunteers below 18 years old and those who abused alcohol and/or heavy smokers were excluded. This study was approved by the Ethics Committee of The First Affiliated Hospital, Sun Yat-sen University. Informed consent was obtained from all subjects enrolled in this study. Clinical characteristics, doppler echocardiographic metrics (the definition and classification of pulmonary hypertension are referenced as in the 2014 Nice Pulmonary Hypertension Classification System) and operation data were collected  . 50 μL o2. After serum starvation, cells were stimulated with 10 ng/mL human PAI-1. Three hours later, the EMPs-rich supernatant was collected and centrifuged  to remove cell debris. The supernatant was removed after ultracentrifugation  and EMPs were resuspended in phosphate-buffered saline (PBS) at room temperature for subsequent experiments.EMPs were generated ex vivo by incubating human umbilical vein endothelial cells (HUVECs) with plasminogen activated inhibitor-1 (PAI-1) as previously described –6. BriefTo confirm the relationship between EMPs and the P38 MAPK pathway, we used targeted siRNAs to reduce expression of P38 MAPK in HUVECs. HUVECs were cultured in endothelial cell medium (ScienCell) supplemented with 5% Fetal bovine serum (FBS), 1% growth factors, and 1% penicillin/streptomycin. Cells were serum starved in 0.5% FBS over night before experiments. Targeted SiRNAs and scrambled siRNAs (negative control)  were transfected into cells with siPORT™ NeoFX™ Transfection Agent  and Opti-MEM I (Gibco). InitialsiRNA concentrations used were 70 nM. At 12 h after transfection, medium was exchanged with endothelial cell medium  consisting of 10% Fetal bovine serum and 10 ng/mL epidermal growth factor to remove siRNA. A separate control group containing only with siPORT™ NeoFX™ Transfection Agent and Opti-MEM I was also employed.5/mL) for 6 h. After centrifuging for 20 min at 12,000×g, the supernatants were collected and tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 levels were evaluated using ELISA kits (ebioscience) as recommended in the manufacturer’s protocol.After siRNA transfection, HUVECs were cultured for an additional 48–72 h until the monolayer of cells reached 90–100% confluence. The cultured cells were then serum starved overnight and stimulated with EMPs  and blood samples were drawn for measurement of pro-inflammatory factors. In addition, the heart was isolated and frozen in liquid nitrogen for further immunoblotting analysis and immunohistochemical staining as previously described [All animal experiments were approved by the Animal Ethics Commission of the First Affiliated Hospital of Sun Yat-sen University. The investigation conformed to the provisions of the Declaration of Helsinki in 1995 (as revised in Edinburgh 2000). Eight-week-old female C57BL6 mice were obtained from the animal center of Sun Yat-sen University, north campus. 1 × 10escribed , 12, 13.The blood samples drawn from mice were centrifuged and plasma was isolated. TNF-α and IL-6 concentrations were determined using ELISA kits (ebioscience).To investigate the effects of EMPs on relevant proteins in the mouse heart, eNOS expression and phosphorylation at Ser1177, P38 MAPK expression and phosphorylation, and expression of caveolin-1 were assessed by immunoblotting with specific antibodies as previously described [5/mL) for 1 h. Then cellular proteins were harvested, total and phosphorylated P38 MAPK, and GAPDH protein expression were determined by immunoblotting as previously described [To investigate the effects of EMPs and siRNAs on cultured cells, HUVECs were evaluated without and with transfection of siRNAs targeting P38 MAPK (and scrambled control siRNAs) as described above. HUVECs were stimulated with EMPs . Anti-eNOS was obtained from Santa Cruz Biotechnology . Anti-GAPDH was obtained from Proteintech Group . Proteins were separated by SDS-PAGE and then visualized using luminol reagent (Santa Cruz Biotechnology) according to standard methods.To investigate the effects of EMPs on relevant protein expression in mouse hearts, isolated mouse hearts treated without or with EMPs were embedded with paraffin, sectioned and subjected to immunostaining. The expression of caveolin-1, eNOS, and P38 MAPK in mouse hearts was detected by immunohistochemical staining using standard protocols. Polycolonal rabbit IgG anti-Caveolin-1 antibody , anti-eNOS antibody  and anti-p38 MAPK antibody  were used as primary antibodies.5 or 5 × 105/mL EMPs or equal volumes of PBS were injected into the mouse tail vein. 6 h later, hearts were isolated and placed in 1:9 (wt/vol) cold homogenization buffer. The hearts were cut into small pieces with an iris scissors and homogenized five times on ice . The homogenates were then centrifuged for 8 min  and the supernatant was isolated. NO concentration was determined by measuring total nitrate plus nitrite (NO3− + NO2−) using an NO detection kit  according to the manufacturer’s instructions. Briefly, nitrate was enzymatically converted into nitrite by nitrate reductase, and nitrite was quantified with Griess reagent at an absorbance of 550 nm, as previously described [1 × 10t test was used. P < 0.05 was considered to indicated statistical significance. Data were presented as mean ± SDEM.Statistical analyses were performed using Prism 5 software. For comparison of healthy subjects, ASD patients, and VSD patients, or the impact of different concentrations of EMPs on protein expression in hearts and cultured cells, one-way ANOVA and Newman-Keuls comparisons were used. Chi-square test was used to compare proportions between different groups. For comparison between EMPs and controls, a Demographic and clinical features are shown in Table Transthoracic echocardiography differences found among the three subject groups are shown in Table When compared with the control group, plasma EMPs levels were significantly elevated in the ASD group and VSD group Fig. A. The hiIn mouse hearts, both immunohistochemical staining and immunoblotting showed that EMPs significantly increased expression of caveolin-1 Figs. A, B; 3A.Both immunohistochemical staining and immunoblotting showed that EMPs dramatically increased P38 MAPK phosphorylation Fig. . EMPs alEMPs significantly stimulated an increase in TNF-α concentration in cultured HUVECs. In EMPs stimulated groups, TNF-α concentration in the group transfected with siRNA targeting P38 MAPK was significantly lower than in cells transfected with the scrambled siRNA control. No differences were observed between control siRNA and cells not treated with siRNA Fig. C. A simi5/mL for 6 h (when compared with controls).As shown in Fig. As shown in Fig. We and others have previously demonstrated that EMPs levels are elevated in patients with valvular heart diseases and this contributes to impairing mitral valve endothelial cell function . We furtThe mechanisms underlying increased numbers of plasma EMPs in ASD and VSD are unclear. Previous studies demonstrated that EMPs can be released from endothelial cell activation and/or apoptosis . The atrPulmonary hypertension is one major complications of congenital heart diseases , 18. In We and others have been demonstrated that EMPs may play an important role in exacerbating endothelial dysfunction –4, 7. ItPerioperative systemic inflammation is one of the major risk factors and complications in cardiac surgery. P38 MAPK signaling mediates myocardial inflammatory responses and endothelial dysfunction that can contribute to myocardial damage following acute injury , 26. P38Study Limitations: Due to the limitations in current technology, we are unable to extract EMPs from patients’ plasma directly to perform animal or cell studies. Instead, in the present study, we have relied on a published strategy to obtain EMPs from cultured endothelial cells stimulated with PAI-1 , 4, 6 thIn summary, our study demonstrated that EMPs were increased in adult patients with congenital heart diseases. These increased EMPs inhibited eNOS activity to reduce NO production and activated P38 MAPK-dependent signaling pathways to promote production of TNF-α and IL-6. These mediators of pro-inflammatory response impaired endothelial function. These changes may contribute to impairment of myocardial and vascular function during the perioperative period to affect hemodynamic stability after cardiac surgery. In addition, the consequences of increased EMPs may also promote development of pulmonary hypertension. We conclude that the P38 MAPK pathway may play a role in EMPs-induced inflammation and provide a potential therapeutic target in surgery of congenial heart disease.


Malaria is the main cause of morbidity and mortality among children under the age of five years in Malawi. The aim of this study was to compare the prevalence and factors associated with malaria parasitaemia among children under the age of five years in Malawi between the 2014 and 2012 Malaria Indicator Surveys (MISs).Data on demographic factors, vector control interventions, and blood for malaria test were collected from a representative sample of children under the age of five years in Malawi through multistage cluster sampling method. Data were analysed by chi-square test and logistic regression using complex samples analysis of the Statistical Package for the Social Sciences (SPSS) version 22.The prevalence of malaria parasitaemia among children under the age of five years increased from 28% in 2012 to 33% in 2014 (p > 0.05). Likewise, the proportion of children using long-lasting insecticide-treated net (LLIN) increased significantly from 54% in 2012 to 65% in 2014 MIS (p < 0.05). The proportion of households that had used indoor residual spraying (IRS) was 9% for both surveys. In multivariate analysis, use of LLIN significantly predicted for malaria parasitaemia in the 2012 MIS but not in the 2014 MIS. Older children and those coming from the poorest families were significantly associated with having malaria parasites in both surveys.The increase in the use of LLIN among children in 2014, did not result in the reduction of malaria parasitaemia in children. The use of LLIN significantly predicted for malaria parasitaemia among children in the 2012 MIS but not in the 2014 MIS. The results of this study underscore the need to increase the coverage of IRS, mosquito repellents and larvicide alongside LLINs in order to reduce the burden of malaria among children in Malawi. Malaria is a leading cause of mortality among children in sub-Saharan Africa . GloballAlthough people from all areas of Malawi are at risk of malaria, those staying in wetter, hotter and more humid, low-lying areas such as along the lakeshore and Shire river valley are at greater risk than those staying in dry and highland areas , 6. In aThe Government of Malawi and international donors are investing a lot of resources towards interventions to reduce the burden of malaria among children under the age of five years , 7. For Despite the Government of Malawi and its development partners increasing resources each year to reduce the burden of malaria in children under the age of five years, little is known as to whether the interventions are well targeted. Without identifying the predictors of malaria parasitaemia in Malawi, government and its donors might be losing the battle in the fight against malaria despite investing a huge amount of resources. This study aimed to evaluate the effectiveness of malaria interventions in Malawi by comparing the prevalence of malaria and its predictors between the 2014 Malaria Indicator Survey (MIS) and 2012 MIS. Not only will this study provide feedback to government and stakeholders on the effectiveness of malaria control interventions, but will also provide a basis for planning of resources and interventions to control the burden of malaria among under five years old children in Malawi.Malawi has a sub-tropical type of climate which favours breeding of anopheles mosquitoes. Breeding of mosquitoes is high in the rainy season, (from November to April) because of the abundance of water, vegetation, and warm weather.Malawi is divided into three administrative regions  and 28 districts . Each district is divided into traditional authorities. For statistical purposes, the 2008 National Population and Housing Census divided these traditional authorities further into standard enumeration areas (SEAs). The 2008 Census also determined the number of households in each area. The 2012 and 2014 Malawi Malaria Indicator Surveys’ sampling frame was the list of SEAs developed from the 2008 Census. The first stage involved stratification of the SEAs by region and residence location . Thereafter 140 SEAs  and 3500 households were selected from the list of 12,569 SEAs from the 2008 census using probability to proportional size. In order to produce robust estimates, the Northern region, and urban areas in the three regions were oversampled making the MIS disproportional to the region and urban population hence needed final weighting adjustment in order to provide reliable estimates for every area of the study. The SEA size was considered as the total number of residential household. The residential households were allocated to the different strata. The second stage involved selection of an average of 25 households from each of the 140 SEAs using systematic sampling from the list of households in the SEAs. Taking into account that it had been several years since the 2008 census was conducted; a fresh household listing was done for both MIS before the commencement of the surveys. New serial numbers were assigned to households during the fresh household listing and were later used to sample participants.Data collection for the 2014 MIS were collected from 2 May 2014 to 10 June 2014 while the 2012 MIS data were collected from April to May 2012. The data collection team consisted of community nurses, laboratory technicians, and the supervisors. The 2014 MIS had a response rate of 99% while the 2012 MIS had a response rate of 98%. The 2014 and 2012 MISs used similar study measures namely; the household, the biomarker, and the questionnaire for women. This paper used data from the household and the biomarker questionnaires. The household questionnaire collected information about all members and visitors of the selected households. The purpose of the questionnaire was to identify eligible women for individual interviews and eligible children aged between six and 59 months to be tested for malaria. The instrument also collected demographic characteristics of the study participants such as age, sex, and the relationship of each household member to the head of the family. In addition, the questionnaire collected information pertaining to dwelling characteristics of the dwelling unit such as toilet facility, the source of water, materials used for the wall, floor and roof of the house, ownership of durable goods as well as ownership and use of the mosquito nets. The socioeconomic status of participants was measured using the wealth index. The wealth index, is a tool developed by the World Bank to measure the household socioeconomic status in developing countries on the basis of household’s ownership of consumer goods, dwelling characteristics, type of drinking water source, toilet facilities, and other characteristics . The bioPlasmodium falciparum.Malaria parasites were detected by microscopy. A thick blood smear was taken from all eligible children and was sent to the laboratory to be microscopically tested for Plasmodium falciparum specific protein rather than the parasite itself hence it is less sensitive than the microscopic test.The outcome variable of the study was testing positive or negative for malaria parasites using microscopic test. Malaria results by microscopy were chosen over rapid test because they are more accurate than those by the latter . Given tThe independent variables were divided into two categories; sociodemographic variables and vector control variables. The sociodemographic variables included the following variables; child’s age, sex, mother’s education, residence, region cluster altitude and household wealth index. Vector control variables consisted of ownership of mosquito net, use of LLIN, and use of IRS.Statistical Package for the Social Sciences (SPSS), IBM version 22 was used to analyse data. MIS used multistage sampling to recruit participants hence it is regarded as having a complex survey design . AnalysiEthical clearance to conduct the study was obtained from Malawi National Health Science Research Committee (NHSRC) prior to data collection. Verbal and written consent were also obtained from the study participants. Parental consent was also sought from all children who were tested blood for malaria parasites. In addition, the authors of this paper sought permission from the International Classification of Functioning, Disability, and Health (ICF), which oversees demographic health survey data including MIS to publish the results of the study.Overall, a total of 4040 children under the age of five years were included in the study, 2112 (52%) from the 2012 MIS and 1928 (48%) from the 2014 MIS data. The prevalence of malaria parasitaemia increased among children under the age of five years from 28% in 2012 to 33% in 2014. Nevertheless, the difference in prevalence of malaria parasitaemia among children between the two surveys was statistically insignificant (p >0.05). As regards to the proportion of households who owned at least one bed net, the 2014 MIS had a significantly higher proportion (77%) compared to the 2012 MIS (70%) (p <0.05). In addition, the proportion of children who slept under LLIN a night before the survey significantly increased from 60% in 2012 to 66% in 2014 (p <0.05). Furthermore, only nine per cent of households had used IRS in the previous 12 months in both surveys (p > 0.05). When the sociodemographic variables in the two surveys were compared, no statistically significant differences were found in the proportions of participants in any of the variables  between the 2012 MIS and 2014 MIS (p > 0.05) see .In bivariate analysis, children from the Central Region, living in rural area, coming from poorest families, older than 11 months, and those whose mothers had lower education level were more likely to have malaria parasites in the 2012 MIS (p < 0.05) see . On the The odds of testing positive for malaria parasites in multivariate analysis in the 2012 MIS were higher among children who did not sleep under the LLIN the night before the survey , children from the Central Region , children living in rural area , children from poorest families , children who were 48 months of age or older  and mothers of children with no education  see . On the The results of this study have shown that the prevalence of malaria parasitaemia among children younger than five years was higher in 2014 compared to 2012 despite a significant increase in the use of LLIN. The findings of this study are not different from those reported in a previous cohort study conducted in Uganda among 100 children who were followed up from six weeks of age until they were 48 months of age . In the In addition, this study has found that not sleeping under LLIN the night preceding the survey was a significant predictor of malaria parasites in children under the age of five years in the 2012 MIS but not in the 2014 MIS. Authors of previous studies have also reported different findings regarding LLIN, with some identifying it as a significant predictor of malaria parasites among children , 22 and This study has also shown that although the use of LLIN increased in 2014, the proportion of households that used IRS did not increase between 2012 and 2014. In 2010, the Government of Malawi and its donors recognising the importance of vector control, initiated the distribution of LLINs and indoors residual spraying (IRS) with pyrethroid insecticide . By 2011In both the 2012 MIS and 2014 MIS, children from poor families were more likely to suffer from malaria compared to those from wealthiest families. There is a great deal of literature, which shows that poverty is a driver of malaria –32. PoorFurthermore, this study has revealed that older children under the age of five years were more likely to test positive for malaria parasites compared to younger children in both the 2012 and 2014 surveys. Normally, the child’s care pattern in Malawi is that younger children (6 to 11 month) are given more priority and are in the total care of their mothers or guardian whereas older children spend more time in the evening playing with siblings before going to bed, and in the process they are bitten by mosquitoes. At night they may move close to the edge of the net and are susceptible to mosquito bites. Younger children in Malawi normally share the same bed with their mothers as such they are properly covered under the mosquito net at night. A study conducted in Uganda showed that children who share a bed with their mother were 21 times more likely to use the mosquito nets compared to those who were not . The finThe results of this study highlight the need for increased efforts to reduce the burden of malaria among children under the age of five years in Malawi. Increasing the coverage of long-acting organophosphate IRS should be one of the government’s priority vector control intervention. IRS can have significant results in the fight against malaria as children can be protected from mosquito bites outside bedtime. A study conducted in Uganda using the MIS data found that the odds of the suffering from malaria were 7.6 times in houses not sprayed against mosquitos as compared to those who were living in houses sprayed against mosquitoes . MoreoveImportantly, considering that children spend a larger part of the day playing outdoors, introducing government or donor-subsidized mosquito repellents can help to protect children from mosquito bites. In rainy season many houses in Malawi, especially in rural areas are surrounded by stagnant water, which provides a potential breeding site for mosquitoes. Larviciding stagnant water around peoples' houses can help to control the population of mosquitoes and so reduce the burden of malaria parasitaemia in Malawi. The results of this study could inform health workers to develop a relevant intervention to address the predictors of malaria among under-five children. When providing health education to primary caregivers, emphasis should be placed on encouraging primary caregivers to ensure that both younger and older children under the age of five years sleep under the LLINs every night. Furthermore, when coming up with policies, guidelines, and interventions to control malaria in children under the age of five years, special attention should be paid to older children and those from poor families.The main strengths of this study are twofold; first, this study used data from two national surveys that had a high response rate of greater than 95%. In addition, the study used survey data collected from a national representative sample as such, the findings can be generalised to children under the age of five years in Malawi. Just like many studies, this study also has limitations. First, the study used cross-sectional design to collect data as such no causal inferences can be made. The second limitation is that we did not include any meteorological data to assess if the climatic variations contributed to the difference in prevalence of malaria parasitaemia. Another limitation of the study is that we did not take into account the seasonal variation in malaria transmission pattern between 2012 and 2014 taking into consideration that the 2014 survey was conducted a month later than the 2012 survey. Future studies should consider assessing the association of climatic variations and malaria parasitaemia in children. Despite these limitations, this study has identified some of the determinants of malaria parasitaemia in children under the age of five years in Malawi that can be used for planning future interventions.The comparison of the two surveys shows that little progress has been made in reducing malaria parasitaemia among children under the age of five years. The increase in the proportion of children who slept under LLIN a night preceding the survey in 2014, did not result in a reduction of malaria parasitaemia in children. Moreover, the proportion of households that used IRS did not increase in 2014. Socio-demographic factors such as older age and coming from poor families were independent predictors of malaria parasitaemia for the two surveys. The results of this study show that use of LLIN alone is not enough to reduce malaria parasitaemia in children in Malawi. The Government of Malawi and international donors should consider increasing the coverage of other interventions such as IRS, mosquito repellent and Larviciding in order to reduce the burden of malaria parasitaemia among children under the age of five years in Malawi. In addition, it is important to take into consideration the socio-demographic factors that are associated with malaria parasites among children in Malawi when planning or implementing vector control interventions in order to have positive outcomes. This study has created a benchmark for the planning of malaria control interventions as well as for comparing progress and successes of future interventions in Malawi.


Plasmodium falciparum and Plasmodium vivax. A better understanding of the acquisition of immunity in naturally-exposed populations is essential for the identification of antigens useful as biomarkers, as well as to inform rational vaccine development.Further reduction in malaria prevalence and its eventual elimination would be greatly facilitated by the development of biomarkers of exposure and/or acquired immunity to malaria, as well as the deployment of effective vaccines against P. falciparum (PfGPI) in a cohort of 1–3 years old Papua New Guinea children with well-characterized individual differences in exposure to P. falciparum and P. vivax blood-stage infections. The relationship between IgG levels to PfGPI and measures of recent and past exposure to P. falciparum and P. vivax infections was investigated, as well as the association between antibody levels and prospective risk of clinical malaria over 16 months of follow-up.ELISA was used to measure total IgG to a synthetic form of glycosylphosphatidylinositol from PfGPI were low in the young children tested. Antibody levels were higher in the presence of P. falciparum or P. vivax infections, but short-lived. High IgG levels were associated with higher risk of P. falciparum malaria , suggesting that they are biomarkers of increased exposure to P. falciparum infections. Given the cross-reactive nature of antibodies to PfGPI, high IgG levels were also associated with reduced risk of P. vivax malaria , indicating that these antibodies are also markers of acquired immunity to P. vivax.Total IgG levels to PfGPI might be a useful marker of immune-status to both P. falciparum and P. vivax infections, and potentially useful to help malaria control programs to identify populations at-risk. Further functional studies are necessary to confirm the potential of PfGPI as a target for vaccine development.This study highlights that in young children, IgG to The online version of this article (doi:10.1186/s12936-017-2042-2) contains supplementary material, which is available to authorized users. Plasmodium falciparum and Plasmodium vivax, would certainly accelerate malaria elimination   , to date[EBA140] –45. Furt malaria .PfGPI were associated with higher risk of P. falciparum malaria. In contrast, they were also associated with reduced risk of P. vivax malaria. This accurately reflects the different levels of naturally acquired immunity to the two species in this cohort: while in these children incidence of P. vivax episodes significantly decreases starting in the 2nd year of life, the burden of P. falciparum infection continues to increase until the 4th year of life [P. vivax than P. falciparum blood-stage infections, i.e. P. vivax molFOB was considerably higher than P. falciparum molFOB . This high number of P. vivax clones that infect children in early childhood thus contribute to a very rapid acquisition of immunity to clinical P. vivax malaria, not yet reached for P. falciparum [P. falciparum in high transmission settings such as PNG is achieved a number of years later (~ 10 years old) with increasing exposure to P. falciparum infections [PfGPI antibodies in this age group seem to be an accurate reflection of the children’s current immune-status to both P. falciparum and P. vivax malaria, acting as both a biomarker of increased risk of P. falciparum, able to identify individuals with the highest level of exposure to P. falciparum recent infections, as well as a biomarker of acquired immunity to P. vivax.In young PNG children, high antibody levels to  of life . This dilciparum , 30. Acqfections , 8. AntiPfGPI were able to delay mortality by Plasmodium berghei, demonstrating proof of concept for a GPI-based anti-toxic malaria vaccine [PfGPIs were shown to be able to block the induction of toxic responses, also suggesting that GPI-based therapy is possible [P. berghei models, the vaccine showed efficacy in sporozoite challenges, was able to reduce parasite replication and transmission to mosquitoes  Altogether, these findings suggest that a GPI vaccine may be able to prevent both blood-stage and liver infections, disease and block transmission of parasite from human to mosquito, thus acting as a unique carbohydrate multi-stage, multi-parasite vaccine. Consistent with this, high levels of the anti-GPI antibodies have been correlated with resistance to clinical symptoms, such as anaemia and fever [PfGPI antibodies are short-lived or intermittent in very young children, older children and adults seem to be able to sustain high antibody levels for longer [PfGPI antibodies contribute to the protection observed against P. vivax, or only act as a mirror of the protection conferred by antibodies to other antigenic targets.In 2002, a study in rodent models firstly showed that antibodies raised against  vaccine . The antpossible , 48. In nd fever , and lownd fever . Althougr longer –34, 50. r longer . Future PfGPI antibodies as a possible biomarker of anti-malaria immunity in very young children. Further studies including older age groups will confirm its utility as a biomarker of immunity for P. vivax, and whether they will indeed also reflect acquired immunity to P. falciparum.This study highlights anti-PfGPI as potentially useful serological biomarkers of immune-status in young children to help malaria control programs identify populations at risk. Additional studies including older age groups will confirm the utility of these responses as a biomarker of immunity to P. vivax, and whether they will indeed also reflect acquired immunity to P. falciparum. Future functional studies are also necessary to confirm whether anti-PfGPI antibodies contribute to the protection observed against P. vivax, or only act as a mirror of the protection conferred by antibodies to other antigenic targets.The findings of this study highlight IgG to Additional file 1. Antibody responses to PfGPI and the BSA tag in 1-3 years old children. Box plots show median IgG levels , minimum and maximum (whiskers) and outliers (grey circles). n=223. P value is from t-test.Additional file 2. IgG to PfGPI by village of residency.Additional file 3. Association between IgG to PfGPI and protection against clinical malaria in Papua New Guinean children.Additional file 4. Influence of age and exposure on antibody levels to PfGPI in Papua New Guinean children.