Plasmodium falciparum merozoite surface protein 3 (MSP3) and glutamate-rich protein (GLURP), in children in their first two years of life in Burkina Faso and their risk of malaria.Children below six months are reported to be less susceptible to clinical malaria. Maternally derived antibodies and foetal haemoglobin are important putative protective factors. We examined antibodies to Plasmodium falciparum antigens MSP3 and two regions of GLURP (R0 and R2) on blood samples collected at baseline, three, six, nine, 12, 18 and 24 months. Foetal haemoglobin and variant haemoglobin fractions were measured at the baseline visit using high pressure liquid chromatography.A cohort of 140 infants aged between four and six weeks was recruited in a stable transmission area of south-western Burkina Faso and monitored for 24 months by active and passive surveillance. Malaria infections were detected by examining blood smears using light microscopy. Enzyme-linked immunosorbent assay was used to quantify total Immunoglobulin G to A total of 79.6% of children experienced one or more episodes of febrile malaria during monitoring. Antibody titres to MSP3 were prospectively associated with an increased risk of malaria while antibody responses to GLURP (R0 and R2) did not alter the risk. Antibody titres to MSP3 were higher among children in areas of high malaria risk. Foetal haemoglobin was associated with delayed first episode of febrile malaria and haemoglobin CC type was associated with reduced incidence of febrile malaria.We did not find any evidence of association between titres of antibodies to MSP3, GLURP-R0 or GLURP-R2 as measured by enzyme-linked immunosorbent assay and early protection against malaria, although anti-MSP3 antibody titres may reflect increased exposure to malaria and therefore greater risk. Foetal haemoglobin was associated with protection against febrile malaria despite the study limitations and its role is therefore worthy further investigation. Children under five years of age bear the majority of the malaria burden This apparent protection has been linked to passive transfer of anti-malaria antibodies by earlier experimental studies Correlates of protection of specific antibodies against malaria have been extensively studied in adults and children P. falciparum malaria in their first years of life. We specifically investigated three merozoite surface proteins, MSP3-LSP and two regions of GLURP (R0 and R2), as they have been used in a malaria vaccine that is currently undergoing testing We performed the present longitudinal prospective infant cohort study in the south-western region of Burkina Faso, where the transmission of malaria is high and stable. Our study aimed at investigating the role of transplacentally acquired anti-malaria antibodies, while adjusting for foetal haemoglobin and other factors, in the susceptibility of children to This study was approved by the Institutional Review Board of Centre National de Recherche et de Formation sur le Paludisme (CNRFP) in Burkina Faso. The study was conducted according to the principles of the Declaration of Helsinki. Individual written informed consent was obtained from the parents of each child before any study procedure was performed. The parents who could not read and write in the language used in the informed consent form, signed the form with their thumb print after it was completed by an independent witness on their behalf. The IRB approved this consent procedure.Plasmodium falciparum being responsible for more than 90% malaria cases.The study area is described elsewhere A cohort of 140 infants aged between four and six weeks was recruited into the study. The parents were informed of the study aims and procedures during the early post-natal visits at the dispensaries, prior to the children reaching one month of age. Recruitment was carried out simultaneously at the four health catchment areas of the study site from November 2010 to February 2011. Since measuring time to the first infection was one of the objectives of the study, infants who had either a documented previous episode of malaria or a positive blood smear at the baseline visit were excluded from the study; nevertheless, some infants might have had malaria infections that were unobserved. After the recruitment of the study participants, the geodetic coordinates  of their homesteads were recorded using Global Positioning System (GPS) devices. The approximate centre of each family compound was the reference point to record these coordinates.To detect malaria infections, the children were followed up actively with weekly home visits and passively with dispensary monitoring for two years. The active follow-up consisted of weekly home visits performed by fieldworkers whose main tasks were to check the children’s health, collect blood samples and start anti-malarial treatment when indicated according to the National Malaria Control Program guidelines. The time window for the home visits was ±2 days. In the passive follow-up, the caregivers were encouraged to bring their children to the nearby dispensary or the clinical research unit at any time should the child appear unwell. In both surveillance methods a blood smear was collected in case of fever (i.e. a reported history of fever in the past 24 hours and/or axillary temperature ≥37.5°C). In addition, to monitor the occurrence of asymptomatic parasitaemia, bloods smears were systematically collected on a monthly basis until the detection of the first malaria infection regardless of the axillary temperature. The children who became ill over the course of the study received health care free of charge either at the dispensaries, the clinical research unit or the paediatric unit of the regional referral hospital when necessary.Light microscopy was used to examine the blood smears, which were collected, air dried and GIEMSA-stained as described elsewhere High Pressure Liquid Chromatography (HPLC) was used to quantify the fractions of foetal haemoglobin and haemoglobin variants in the children.P. falciparum blood-stage antigens MSP3-LSP Enzyme-linked immunosorbent assay (ELISA) was used to quantify anti-malaria total IgG in capillary blood samples collected by finger or heel prick at baseline, three, six, nine, 12, 18 and 24 months of follow up. Two P. falciparum parasitaemia, and definition 2 included only febrile episodes associated with asexual P. falciparum parasite density ≥10000/µL. This latter definition was derived after examining the distributions of the log-transformed parasite densities among children with and without fever in cross-sectional surveys  to our cohort with time-to-event data We fitted a Cox regression model to estimate the relationship between the time to first febrile malaria episode and a set of covariates of interest. We also fitted a negative binomial regression model to estimate the association between multiple malaria episodes and a set of potential explanatory covariates. Antibodies were fit in two ways; a) applying the baseline antibody titre throughout the period of monitoring and b) applying time-varying antibody titre, i.e. the antibody titre measured at the most recent time point, which therefore changed throughout the period of monitoring. The log likelihood ratio test was used to test the significance of variables with multiple levels. The assumption of proportional hazards for Cox regression was tested based on the Kaplan Meier method and the Schoenfeld residuals. The assumption of a linear effect of antibody titres on malaria episodes was investigated using fractional polynomial modelling. We used the Huber-White Sandwich estimator to adjust for clustering by individual in linear and negative binomial regression models. The data were analysed using Stata 13 .A total of 216 mothers of new-borns were invited to attend study screening visits during early post-natal visits at the four dispensaries of the study area. Of these, 148 (68.5%) attended the study screening visits with their infants. A total of 140 infants were recruited during a three- month period from mid-November 2010 to mid-February 2011. The baseline characteristics of the infants and their mothers are summarised in Twenty-three children (16.4%) were lost to follow-up before completing 24 months with a median [Inter Quartile Range-IQR] follow up time of 9.8  months. Among them, six migrated out of the study area, 10 withdrew their consent, four died and three dropped out of the study and were no longer reachable.One or more episodes of febrile malaria were experienced by 79.6% of all children during follow-up. Sixty-three children (45.98%) had at least one malaria infection in their first year of life and 46 (76.66%) of the remaining 60 children in their second year.p<0.001) than either GLURP sub-unit antibody was correlated to anti-MSP3 antibody titres .Antibody titres from one time point were weakly to moderately correlated with antibodies at the next time point  with the strongest correlations observed from baseline to month 3, and months 9 to 12 . The timThe univariate analysis of all potential covariates is displayed in p = 0.003, 95% CI ) which was not significant in univariate analysis (p = 0.064). Belonging to the haemoglobin CC type group was also significantly associated with decreased malaria incidence  in the multivariable but not in the univariate analysis.Anti-MSP3 antibody titres were significantly associated with an increased hazard of the first febrile malaria episode on univariate  and multanalysis . This wap = 0.082, in the direction of decreasing hazard over time) and for foetal haemoglobin .We did not detect significant variation in the proportionality of hazards over time , but theThe results of negative binomial regression were consistent with the Cox regression analysis . The besRestricting the observation period to six weeks post bleeding for plasma, to take into account the fact that anti-malaria antibodies are more likely to be short-lived in young children In this study, transplacentally acquired anti-malaria antibodies and foetal haemoglobin were investigated in relation to susceptibility to malaria in a cohort of 140 infants. Antibody titres to GLURP and MSP3 were found to decline in the first four months of life, presumably due to the loss of maternal antibodies. Endogenous production was responsible for the subsequent increase in the case of GLURP and stabilisation of the loss of antibodies for MSP3. Contrary to our expectations, there was no association between antibody titres to GLURP (R0 and R2) and the risk of febrile malaria in the first two years of life; antibody titres to MSP3 even appeared as a marker of exposure since it was statistically significantly positively associated with the incidence of febrile malaria and inversely associated with time to first febrile malaria episode. Confounding between malaria risk and antibody titre by variation in exposure has previously been reported P. falciparum malaria, most of the previous studies did not specifically target infants. A number of prospective sero-epidemiological studies have investigated the role of antibodies to these merozoite surface antigens in older children and adults in West Africa, East Africa as well as South-East Asia.In the investigation of the role of antibodies to MSP3 and GLURP (R0 and R2) against The lack of association between antibodies to GLURP (R0 and R2) and protection against clinical malaria in our study is partially concordant with the findings of a previous study in older children in Burkina Faso. Nebie and colleagues investigated total IgG to GLURP (R0 and R2) among others antigens in children aged 6 months to 10 years and found a protective effect for antibodies to GLURP R0 but not to GLURP R2 when the antibody titres were analysed individually. When antibody titres to all the four antigens studied  were included in a multivariable model, antibodies to GLURP (R0 and R2) were not associated with protection against malaria Antibodies to MSP3 have been associated with protection in previous sero-epidemiological studies P. falciparum malaria antigens have been investigated. In sero-epidemiological newborn cohort studies, antibodies to crude P. falciparum schizont extract and MSP2 were found to be associated with higher risk of malaria infection in infants Although antibodies to MSP3 and GLURP have not been previously studied in newborn cohorts, antibodies to other Antibody titres to MSP3 and GLURP (R0 and R2) were not associated with protection in our study, and we suggest that confounding due to exposure led to an apparent association with increased susceptibility for antibody response to MSP3. Limitations of our study include the fact that the high malaria transmission season began 5–7 months after recruitment. Therefore the majority of the maternal antibodies were likely gone by the time that febrile malaria episodes began, and children were exposed during a period of lower antibody titres. We did not use an external control to quantify malaria antibodies as performed elsewhere Interestingly, foetal haemoglobin was significantly inversely associated to febrile malaria incidence although the effect size was relatively small. The effect was only statistically significant on multivariable analysis, and appeared to depend on adjusting by exposure index in the multivariable model. Furthermore the effect seems to be evident after 6 months of age, when we would expect foetal haemoglobin to have been lost from the circulation. We speculate that an interaction between malaria exposure and foetal haemoglobin may be responsible for a delayed protective effect, perhaps due to an early but controlled infection in the presence of high levels of foetal haemoglobin leading to more rapid acquisition of immunity Children who carried the haemoglobin CC type appeared to have a significantly lower risk of malaria as compared to haemoglobin AA type children, as has been previously reported P. falciparum febrile malaria in children in their first few months of life. Despite the above mentioned limitations of the study, the baseline fraction of foetal was associated with protection against febrile malaria. Its role in the protection of children against malaria in their first few months of life is therefore worthy of further investigation. Finally, our study also underlines that the role of haemoglobinopathies should be taken into account in the exploration of protective factors in the low susceptibility of infants to malaria.In conclusion, the present study did not find any evidence of association between antibody titres to MSP3 and GLURP (R0 and R2) and protection against We are grateful to the parents/guardians of the children in Banfora whose commitment and patience made this study possible. We thank the fieldworkers’ team and nurses in the dispensaries who monitored these children for two years as well as all the lab staff. We would also like to especially thank Dr Michael Theisen who kindly provided the malaria antigens used in this study, MSP3-LSP, GLURP R0 and GLURP R2.Figure S1Distribution of parasitemia in febrile and afebrile children for the whole study period. The box-and-whisker plot represents the median and the inter-quartile range of the parasite density in a log 10 scale in the febrile and afebrile children groups.(TIF)Click here for additional data file.Figure S2Selection of the best radius for individual malaria exposure index calculation. The lowest log likelihood in the univariate Cox regression analysis was the selection criteria for the radius to be used.(TIF)Click here for additional data file.Figure S3Dynamics of total IgG to MSP3 over the whole study period using a 25% random sample representing 23 children. Each line represents a child. The antibody titres are in log 10 scale.(TIF)Click here for additional data file.Figure S4Test of proportional hazards assumption: Schoenfeld residuals plots. (A) Baseline total IgG to MSP3, (B) Baseline total IgG to GLURP R0, (C) Baseline total IgG to GLURP R2, (D) Changing total IgG to MSP3, (E) Changing total IgG to GLURP R0, (F) Changing total IgG to GLURP R2, (G) Individual malaria exposure index, (H) Baseline Foetal haemoglobin rate.(TIF)Click here for additional data file.Figure S5Scatter plot matrix of the continuous independent variables used used in multivariable regression models.(TIF)Click here for additional data file.Figure S6Multivariable fractional polynomial plots for antibodies to MSP3 (A), GLURP R0 (B) and GLURP R2 (C).(TIF)Click here for additional data file.Table S1Variability of antibody titres dynamics. The correlations (r) are examined between every two consecutive time points for antibody measurement.(DOC)Click here for additional data file.Table S2Predictive model for changing anti-malaria antibody titers using linear regression. Univariate analysis.(DOCX)Click here for additional data file.Table S3Cox regression analysis using changing antibody titres.(DOCX)Click here for additional data file.Table S4Predictive model for the occurrence of febrile malaria episodes using changing antibodytitres.(DOCX)Click here for additional data file.Table S5Predictive model for the occurrence of febrile malaria episodes using changing antibody titres with a restricted observation period (6 weeks) post-bleeding for plasma.(DOCX)Click here for additional data file.Table S6Multicollinearity diagnostics for continuous independent variables used in multivariable regression models.(DOCX)Click here for additional data file.Table S7Testing for interactions between predictors in multivariable negative binomial regression model.(DOCX)Click here for additional data file.


Good house construction may reduce the risk of malaria by limiting the entry of mosquito vectors. We assessed how house design may affect mosquito house entry and malaria risk in Uganda.100 households were enrolled in each of three sub-counties: Walukuba, Jinja district; Kihihi, Kanungu district; and Nagongera, Tororo district. CDC light trap collections of mosquitoes were done monthly in all homes. All children aged six months to ten years (n = 878) were followed prospectively for a total of 24 months to measure parasite prevalence every three months and malaria incidence. Homes were classified as modern  or traditional .Anopheles were collected over 6,765 nights. 6,816 routine blood smears were taken of which 1,061 (15.6%) were malaria parasite positive. 2,582 episodes of uncomplicated malaria were diagnosed after 1,569 person years of follow-up, giving an overall incidence of 1.6 episodes per person year at risk. The human biting rate was lower in modern homes than in traditional homes (adjusted incidence rate ratio (IRR) 0.48, 95% confidence interval (CI) 0.37–0.64, p<0.001). The odds of malaria infection were lower in modern homes across all the sub-counties , while malaria incidence was lower in modern homes in Kihihi  but not in Walukuba or Nagongera.A total of 113,618 female House design is likely to explain some of the heterogeneity of malaria transmission in Uganda and represents a promising target for future interventions, even in highly endemic areas. The population of Africa is expected to double to nearly two billion between 2010 and 2040 and may reach three billion by 2070 ,6 13]..13].Anopheles gambiae s.l., the major African malaria vector, enters houses at night through open eaves, the gap between the top of the wall and the roof  KR of 32.  The prims. (99%) . Nagonge of 310.  The primis (19%) . Rainfalis (19%) Before the start of the study, a census was conducted in all three sub-counties and a random sample of households selected for screening. From August 2011 to September 2011, children from 100 households randomly selected from the census survey were enrolled into a cohort study and followed for 24 months until September 2013 if they met the following eligibility criteria: (1) aged six months to less than 10 years, (2) resident of the household selected for recruitment, (3) no intention to move out of the sub-county for the next two years, (4) agreement to attend the study clinic for any febrile illness, (5) agreement to avoid antimalarial medications administered elsewhere and (6) provision of written informed consent. Recruitment was dynamic such that children reaching six months of age and meeting the eligibility criteria were enrolled, and children were withdrawn when they reached eleven years of age. The sample size of 300 children for each site was calculated for a separate study comparing temporal changes in malaria incidence from the cohort studies with temporal changes in malaria test positivity rate from health facility based surveillance. The analysis described here is a secondary analysis making use of these data sets. The study data are available in At enrolment, a baseline clinical evaluation was conducted and study participants were given a LLIN . Parents of participants were requested that their children attend the designated study clinic, open seven days a week, for all healthcare needs. Subjects presenting with a fever or history of fever within the past 24 hours with a positive blood smear were diagnosed with malaria. Episodes of uncomplicated malaria were treated with artemether-lumefantrine and complicated episodes treated with quinine. New episodes of malaria were diagnosed by passive case detection and malaria episodes defined as any treatment for malaria. Routine visits were conducted at the study clinic every three months, with a standard evaluation including a thick blood smear to assess for parasitaemia.Thick and thin blood smears were stained with 2% Giemsa and read blind. Blood smears were considered negative when the examination of 100 high power fields did not reveal asexual parasites. All slides were read twice and discrepancies resolved by a third reviewer. In addition, all positive blood smears with a parasite densities ≤20,000/μl based on the field readings were re-read by an expert microscopist based in Kampala and had to be confirmed to be considered positive in the final analyses.Detailed descriptions of the entomological studies are provided elsewhere . In brieEach household was visited at baseline and a questionnaire administered to the head of the household to record data on features of the house , which were independently validated by field assistants, together with household demographics and proxy wealth indicators.Data were collected using a paperless system for the household survey and using standardized case record forms entered into Microsoft Access for follow-up of study participants. Analyses were performed with Stata Version 13 . Missing data were excluded from the analysis.Principal component analysis (PCA) was used to create a wealth index from 10 factors : ownershAnopheles caught per house by light trap catches, with the number of sampling nights included as an offset term in the model. The odds of malaria infection at the time of each routine clinic visit was modelled using logistic regression and negative binomial regression used to model the number of malaria cases per child. Robust standard errors were used to adjust for clustering due to household in both models. We estimated an incidence rate ratio (IRR) for the association between house type and human biting rate (HBR) adjusted for household wealth; and an odds ratio and incidence rate ratio for the association between house type and malaria adjusted for age, gender and household wealth. The associations were analysed separately for each study site and Wald tests were used to test for effect modification by study site.Main wall material, main roof material and eave type were used to classify homes as either modern  or traditional . Negative binomial regression was used to model the relationship between household risk factors and the number of Written informed consent was obtained in the appropriate language from guardians for the participation of their child and from an adult household member for the light trap catches and household surveys. Approval from local leaders was obtained before beginning activities. Ethics approval was provided by the Uganda National Council for Science and Technology; Makerere University School of Medicine Research and Ethics Committee; University of California, San Francisco Committee for Human Research; and the London School of Hygiene and Tropical Medicine Ethics Committee.In total 878 children were enrolled; 251 in Walukuba, 327 in Kihihi and 300 in Nagongera ; Table 1The first principal component explained 21.5% of the overall variability in the asset variables. The weight assigned to each variable was: radio ownership (0.43), table ownership (0.41), cupboard ownership (0.39), mobile ownership (0.34); frequency of problems satisfying food needs (0.33), toilet access (0.33), clock ownership (0.32), bicycle ownership (0.18), main mode of transport to health facility (0.14), and meat consumption (0.12).Anopheles were collected over 6,765 nights of collection. Data were missing for one household. Overall, HBR was highest in Nagongera  and lower in Kihihi (4.6) and Walukuba (1.1). In Kihihi and Nagongera, HBR was lower in homes with tiled or metal roofs and homes with cement, wood or metal walls  and lower in Kihihi (9.4%) and Walukuba (7.4%). The association between house type and odds of malaria infection varied by site (p<0.001). Controlling for age, gender and household wealth, the odds of malaria infection were lower in children living in modern homes than in traditional homes in Walukuba (Odds Ratio (OR) 0.35, 95%CI 0.13–0.92, p = 0.03), Kihihi  and Nagongera   were positive. All children contributed at least one routine blood smear.  = 0.01) . Overall2,582 episodes of uncomplicated malaria were diagnosed after 1,569 person years of follow-up, yielding an overall incidence of 1.6 episodes per person year at risk (PPY). Five participants were withdrawn immediately after screening and did not contribute person years at risk. Incidence was highest in Nagongera (2.8 episodes PPY) and lower in Kihihi (1.4) and Walukuba (0.4). The association between house type and malaria incidence varied by site (p = <0.001). Controlling for age, gender and household wealth, malaria incidence was 39% lower in children living in modern homes in Kihihi  but not in Walukuba or Nagongera .We investigated the association between house construction and malaria at three sites in Uganda: peri-urban Walukuba with low malaria transmission, rural Kihihi with moderate transmission and rural Nagongera with high transmission. Modern homes were associated with a 52% reduction in HBR after controlling for site and household wealth. Similarly, the odds of malaria infection were 56% lower in children living in modern homes than those living in traditional homes, after controlling for age, gender, site and household wealth. These results show that reducing vector biting rates by half is associated with a similar proportional reduction in malaria infection. A similar result was found in a randomised controlled trial of house screening in The Gambia which showed that house screening reduced malaria transmission by half, with a similar reduction in the risk of malaria anaemia .An. gambiae. This vector follows human odour plumes until it reaches an external house wall, flies upwards and, funnelled by the inclined roof, enters the house through open eaves An. gambiAn. gambiae s.s., a highly endophagic vector. In contrast, no association was observed in Walukuba, where 64% of malaria vectors are the less endophagic An. arabiensis, nor in Nagongera, where 19% vectors are An. arabiensis. Screening interventions also may not be as effective against culicine mosquitoes as An. gambiae, reducing their potential appeal to homeowners. Moreover, restricting air flow in homes may increase the internal temperature and the risk from respiratory diseases, especially if wood is burned indoors [Reducing the number of entry points into a house is not a panacea. Most obviously, interventions built into the home do not protect against outdoor transmission . We obse indoors . HoweverOur findings may also not be generalisible to other countries with different house styles and vector ecology. Furthermore, the observed association between house type and malaria risk is not evidence of causality. Indeed, since the direct and indirect costs of malaria can contribute to poverty within a household , especiaWe provide evidence that house structure may explain some of the often marked heterogeneity of transmission in Uganda. Improving house design should be further evaluated as a potential malaria control intervention in sub-Saharan Africa, even in areas of very high transmission.S1 Data.(ZIP)Click here for additional data file.


Malaria is one of the leading causes of acute febrile illness (AFI) in Africa. With the advent of malaria rapid diagnostic tests, misdiagnosis and co-morbidity with other diseases has been highlighted by an increasing number of studies. Although arboviral infections and malaria are both vector-borne diseases and often have an overlapping geographic distribution in sub-Saharan Africa, information about their incidence rates and concurrent infections is scarce.From July 2009 to March 2013 patients from seven healthcare facilities of the Kedougou region presenting with AFI were enrolled and tested for malaria and arboviral infections, i.e., yellow fever (YFV), West Nile (WNV), dengue (DENV), chikungunya (CHIKV), Crimean Congo haemorrhagic fever (CCHFV), Zika (ZIKV), and Rift Valley fever viruses (RVFV). Malaria parasite infections were investigated using thick blood smear (TBS) and rapid diagnostics tests (RDT) while arbovirus infections were tested by IgM antibody detection (ELISA) and RT-PCR assays. Data analysis of single or concurrent malaria and arbovirus was performed using R software.A total of 13,845 patients, including 7387 with malaria and 41 with acute arbovirus infections  were enrolled. Among the arbovirus-infected patients, 48.7 % (20/41) were co-infected with malaria parasites at the following frequencies: CHIKV 18.7 % (3/16), YFV 58.3 % (7/12), ZIKV 88.9 % (8/9), DENV 33.3 % (1/3), and RVF 100 % (1/1). Fever ≥40 °C was the only sign or symptom significantly associated with dual malaria parasite/arbovirus infection.Concurrent malaria parasite and arbovirus infections were detected in the Kedougou region from 2009 to 2013 and need to be further documented, including among asymptomatic individuals, to assess its epidemiological and clinical impact. Arboviral infections and malaria are acute vector-borne diseases and concurrent infections are observed , 2, espeIn Senegal, the introduction of malaria rapid diagnostics tests (RDT) in 2007 showed that the prevalence of malaria among acute febrile illnesses (AFI) was largely overestimated while other infections, such as bacteria and arbovirus illnesses were under-reported , 18. In The study was conducted in the Kedougou region, southeastern Senegal Fig.  with an Patients presenting with AFI were recruited from seven healthcare facilities of the Kedougou region, including Ninefesha rural hospital, Kedougou and Saraya health centres, Bandafassi and Khossanto health posts, the Kedougou military health post, and the Catholic Mission mobile team, which targets populations in remote areas Fig. . AFI wasHuman sera were systematically tested for malaria parasite and arbovirus infections. The malaria diagnosis procedure was performed using thick blood smear (TBS) and an RDT  as previously described , 22, whiData were analysed using STATA  and R soThe study objectives, benefits and risks were explained in the French language or local dialects to all participants before inclusion. Written informed consent was obtained from all adult participants and from parents or legal guardians of children. The study was examined and approved by the Senegalese National Health Research Committee.From July 2009 to March 2013, 13,845 patients with AFI  were enrolled from the following seven health facilities: Ninefesha rural hospital (13.5 %), the Kedougou health centre (13.0 %), Saraya health centre (4.8 %), Bandafassi health post (9.7 %), the Military health post (53.8 %), Khossanto health post (3.6 %), and the Catholic mission mobile team (1.4 %). The median age was 16 years, ranged from 1 to 90 years, and the male/female sex ratio was 1:4. Among enrolled patients, 7387 (53.4 %) were malaria-confirmed cases (median age 13 years (range 1–90); male/female sex ratio 1:35), and 41 (0.3 %) were arboviral-infected individuals i.e., 12 YFV, nine ZIKV, one RVF, 16 CHIKV, and three DENV . No patients tested positive for WNV or CCHF. The distribution of positivity for IgM and RT-PCR are summarized in Table As shown in Fig. Among patients infected with arboviruses, 48.7 % 20/41) were concurrently harbouring positive testing for malaria parasites. Distinct differences in co-infection rates were observed among the tested arboviruses. Malaria co-infection was identified in 18.7 % 3/16) of CHIKV, 58.3 % (7/12) of YFV, 88.9 % (8/9) of ZIKV, 33.3 % (1/3) of DENV, and 100 % (1/1) RVF patients. These co-infection rates were not significantly different from one another except between CHIKV and ZIKV . Given the prevalence of malaria and the circulation of distinct arboviruses in this region, these observed co-infection rates reached expected levels, with the exception of CHIKV where co-infection was significantly lower than expected . Patients concurrently infected with malaria parasites and arboviruses were significantly younger, with median age of 14 years compared to 29 years in non-co-infected patients . Among co-infected patients, 67 % presented high grade fever (temperature ≥40 °C), while only ten and 34 % of patients, respectively, were diagnosed with a single arboviral or malaria infection with temperature ≥40 °C . Further, high-grade fever (≥40 °C) was significantly associated with patients exhibiting dual infection of malaria parasites and arbovirus compared to patients with single malaria attack. Although P. vivax  were repArboviral infections and malaria are known to be endemic in the Kedougou region , 14, allAnopheles funestus, a major malaria vector in southeastern Senegal and elsewhere in Africa [A. coustani, which were found infected with CHIKV, YFV and ZIKV in Kedougou region . When considering the high number of asymptomatic malaria and arboviral infections in endemic regions where both vectors co-exist [Another mechanism by which a patient may become infected by both co-exist , 35, 36,Given the similar clinical presentation of arboviral infections and malaria, and the lack of pathognomonic signs and symptoms for any of the diseases, it is difficult to determine which pathogen was responsible for the clinical signs and symptoms in the concurrent infections. For instance, all patients dually infected with malaria parasites and arboviruses had relatively mild and/or non-specific syndromes, including fever, headache, myalgia, body pain, and vomiting. In addition, the fact that arboviral infections are considered by healthcare workers only if malaria tests are negative, sets the stage for the misdiagnosis and under-reporting of concurrent infections. A significant number of the co-infected patients in this study exhibited a fever ≥40 °C compared to patients with malaria or arboviral Table  infectioOne the limitation of the present study is that all the enrolled individuals were febrile patients. However, given that malaria parasites as well as arboviruses can be detected using molecular tools  in asympPlasmodium spp. and arboviruses are frequent in Kedougou where competent vectors of both diseases are abundant. Vector competence and co-infection of certain malaria vectors, also regularly found infected by arboviruses, deserves further investigation. The frequent detection of arboviral disease outbreaks in the Kedougou region highlights the need to strengthen surveillance of AFI for a better estimation of human impact of arboviruses, as well as morbidity and mortality associated with concurrent malaria and arboviral infections. Finally, the high-grade fever ≥40 °C suggests the possibility of malaria and arboviral infection and should help to establish prompt and better care of individuals.This study showed that co-infections between


Plasmodium falciparum and the acquisition of immunity in children at the extreme ends of the over-dispersed distribution of the incidence of clinical malaria.Epidemiological studies indicate that some children experience many more episodes of clinical malaria than their age mates in a given location. Whether this is as a result of the micro-heterogeneity of malaria transmission with some children effectively getting more exposure to infectious mosquitoes than others, or reflects a failure in the acquisition of immunity needs to be elucidated. Here, we investigated the determinants of increased susceptibility to clinical malaria by comparing the intensity of exposure to n = 35), remained malaria-free (n = 12) or had a single episode (n = 26). We quantified antibodies against seven Plasmodium falciparum merozoite antigens in plasma obtained at six cross-sectional surveys spanning these five years. We analyzed the antibody responses to identify temporal dynamics that associate with disease susceptibility.The study was nested within a larger cohort in an area where the intensity of malaria transmission was low. We identified children who over a five-year period experienced 5 to 16 clinical malaria episodes  and had a significantly higher malaria exposure index, than those in the malaria-free or single episode groups . In contrast, the five-year temporal dynamics of anti-merozoite antibodies were similar in the three groups. Importantly in all groups, antibody levels were below the threshold concentrations previously observed to be correlated with protective immunity.Children experiencing multiple episodes of malaria were more likely to be parasite positive by microscopy at cross-sectional surveys  contains supplementary material, which is available to authorized users. Plasmodium falciparum malaria in malaria-endemic areas has long been recognized as a common feature of the epidemiology of malaria [Heterogeneity in the risk of  malaria . Recentl malaria , Uganda  malaria  and Keny malaria ,5 as wel malaria . In Sene malaria . Analysi malaria .P. falciparum antigens is unknown.The factors underlying the heterogeneous epidemiology of malaria are not fully understood. The heterogeneity has been partly attributed to differences in: human genetic  and behaHere, we describe the temporal dynamics of anti-merozoite antibodies in children who were part of the Kenyan cohort described above  and diffThe study was conducted within a longitudinally monitored population in Ngerenya, located within Kilifi District at the Kenyan coast ,12. ThisThe malaria exposure index estimates a distance-weighted local prevalence of malaria infection within a kilometer radius around an index child . In esseP. falciparum in whole blood samples collected at the cross-sectional surveys has been described previously [The detection of eviously . BrieflyP. falciparum msp2 gene was performed as previously described [msp2 domain, followed by nested reactions with fluorescently labeled primers targeting the FC27 and IC-1/3D7allelic types of msp2. Fragment sizes were determined by capillary electrophoresis and analyzed using GeneMapper software (Applied Biosystems).Genomic DNA was extracted from packed erythrocytes using QiaAmp Blood Mini kit . Genotyping of escribed . BrieflyEscherichia coli. The 19-kilodalton fragment of merozoite surface protein (MSP) one-1 (MSP-119) [P. falciparum reticulocyte-binding homologue 2 (PfRh2) [msp2 allelic family) and MSP-2_CH150/9  [Pichia pastoris as 6xHis-fusion proteins [PfRh2 and MSP-119, all antigens were expressed as full-length proteins.Five recombinant antigens representing four vaccine candidate antigens were expressed in MSP-119) , P. falc family)  were exp family) . Apical proteins  and geneP. falciparum unexposed donors residing in the United Kingdom, were included in each plate to allow for the determination of seropositivity cut-offs. The seropositivity cut-off was determined as the mean fluorescent intensity (MFI) of the negative control plus two standard deviations.Plasma immunoglobulin G (IgG) to the recombinant antigens was measured using a previously described multiplex bead-based assay . Seriall19, MSP-2, MSP-3_3D7 and AMA-1 antigens were 59, 19, 16 and 55 AUs, respectively [PfPR2–10 of 44% [PfPR2–10 of 49% [fPR2–10 is a measure of malaria transmission intensity at a population level [Data analysis was performed using STATA 11.2. Antibody titers measured in this study were compared to threshold antibody concentrations. These are antibody concentrations against individual antigens measured that appeared to be associated with protection against clinical episodes of malaria in two independent cohort studies ,15, and ectively ,15. Anti0 of 44%  and a co0 of 49% . The PfPon level -28.Differences in rates of change in antibody titers over the study period were tested using a multilevel mixed-effects linear regression model that accounts for inherent correlations between repeated measurements done on the same subject . In thisP. falciparum transmission was highest  and were classified into the malaria-free group  were excluded from the present analysis so as to allow for a comparison of antibody responses in children at the extreme ends of the over-dispersed clinical malaria incidence distribution , antibody titers to all antigens were comparable among the three study groups. Univariate analysis showed evidence for a significantly higher rate of increase in antibody titers to AMA-1_FVO and AMA_3D7 over the five-year period in the multiple-episodes compared to the single-episode group of children Table . After aPlasmodium falciparum merozoite antigens were generally similar in all groups of children. Of note, antibody levels in all groups of children were lower than those previously shown to correlate with protection against clinical malaria. These data suggest that the differences in susceptibility to clinical malaria among children in this context are attributable to differences in exposure to infectious mosquitoes rather than to a failure to acquire immunity, as reflected by anti-merozoite antibodies.This is, to our knowledge, the first attempt to investigate the underlying immunological determinants of the over-dispersion of clinical episodes of malaria in a low transmission intensity setting. Our study showed that over a five-year period, children who were susceptible to multiple clinical attacks of malaria had higher indices of exposure to infectious mosquitoes than those with single or no episodes. In contrast, the temporal dynamics of antibody responses to msp2 genotypes) at the cross-sectional surveys is a strong indication that children in the multiple episodes groups are indeed more exposed to the parasite. In the absence of clear differences in the acquisition of immunity (as estimated by anti-merozoite antibodies), our data suggest that the differences in disease susceptibility were driven by the observed differences in the intensity of exposure to the parasite.The malaria exposure index  estimateP. falciparum exposure (as estimated by PfPR) in the three groups of children studied here did not exceed 30% at any of the cross-sectional surveys over five years. This is relatively low compared to the 40% cutoff above which a population is considered to be under high malaria transmission [Malaria transmission intensity was low in the study area. smission , and is smission ,15. It mOverall, with the exception of AMA-1-FVO, the rate of change of antibody titers over time did not differ significantly between the three groups. The absence of any significant differences in the temporal change in antibody titers between the groups could be attributable to the young age of the study subjects, small sample size or the antigens studied. It is possible that the young age of the study subjects precludes any potential differences in rates of buildup of antibodies among the study groups. This is plausible considering that, given uniform parasite exposure, the buildup of antibody titers is slower in children than in adults , implyinP. falciparum infections. Considering that differentiation of MBCs into SLPCs peaks six to eight days after re-exposure to antigen [msp2 genotype to the infection could not be determined using capillary sequencing as employed here, interpretation on disease causation and/or immunity should be cautious.Generally, we observed allele-specific boosting of anti-MSP-2 antibodies, which has also been reported in relation to MSP-2  and AMA- antigen , antibod antigen . The incThe observed difference in the rates of change of antibody titers to AMA-1-FVO, but not to the other antigens in the three study groups, may be a reflection of antigen-specific differences in human immune responses to different antigens. Antibody longevity  and affiWe observed that children in the multiple-episodes group had more genetically-diverse infections compared to children in the single-episode and malaria-free groups. This finding is consistent with previous observations that genetically-diverse infections in children are associated with an increased risk of malaria ,41,42. WP. falciparum exposure on antibody titers in the children studied here [In our study, other than at cross-sectional surveys, children were only tested for malaria parasites when they were symptomatic. Thus, it is possible that some asymptomatic infections were undetected, but that would apply equally to all groups and thus is unlikely to introduce bias. Another limitation of the study was the availability of antibody measurements only at the cross-sectional surveys and not when children were symptomatic. Thus, we could not compare antibody levels at the point of symptomatic infections in the multiple- and single-episode groups. We were also unable to account for the possible effects of HIV, malnutrition and prenatal ied here .In summary, our data shows that in the context of a low malaria transmission setting, multiple episodes of clinical malaria are more likely the consequence of increased exposure rather than failure to acquire immunity. We hypothesize that intensive exposure induces antibodies at protective concentrations, while little to modest exposure may manifest as multiple clinical infections with low levels of antibodies. Future studies on the determinants of increased susceptibility to clinical malaria in areas with high malaria transmission intensity will complement the data presented here and help to define the tipping point in malaria transmission intensity where exposure translates into protective immunity as opposed to increased susceptibility to disease. Our data have implications for interventions including vaccines that effectively lower but do not completely abolish malaria transmission intensity. These interventions may reduce malaria transmission intensity below the threshold necessary to induce protective immunity and thus drive increased susceptibility to clinical malaria.


Symptoms caused by bacterial, viral and malarial infections usually overlap and aetiologic diagnosis is difficult. Patient management in low-resource countries with limited laboratory services has been based predominantly on clinical evaluation and syndromic approaches. However, such clinical assessment has limited accuracy both for identifying the likely aetiological cause and for the early recognition of patients who will progress to serious or fatal disease. Plasma-detectable biomarkers that rapidly and accurately diagnose severe infectious diseases could reduce morbidity and decrease the unnecessary use of usually scarce therapeutic drugs. The discovery of microRNAs (miRNAs) has opened exciting new avenues to identify blood biomarkers of organ-specific injury. This review assesses current knowledge on the relationship between malaria disease and miRNAs, and evaluates how future research might lead to the use of these small molecules for identifying patients with severe malaria disease and facilitate treatment decisions. The burden of infectious diseases disproportionately affects the African continent, where massive diagnostic needs have to be met with limited resources. Considerable advances have been made in the management of African patients with febrile illness and in ensuring that those who are severely ill can have access to hospital . HoweverPneumonia and malaria are two leading causes of morbidity and mortality in children aged under 5 years worldwide , especiaPlasmodium infection is confirmed, causal attribution of symptomatology to malaria is not straightforward, as malaria-positive slides are a common incidental finding in areas where many people are semi-immune to malaria and have high rates of asymptomatic parasite carriage  or glycolytic enzymes  with a working threshold of approximately 100 parasites/µL  or protein complexes, including argonaute 2 . The mecA good biomarker has to be specific, with high sensitivity and able to make accurate, reliable and reproducible diagnostic predictions. Also, they have to be robust and as little invasive as possible. miRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology, and to reflect disease status as well as organ damage and injury . As secrThere are more than 1000 studies about miRNAs as biomarkers in human diseases, most of them in different types of cancer. Carcinogen cells actively secrete specific miRNAs into the circulation, sometimes supporting the growth and progression of the tumour . Given thttp://bioeng.swjtu.edu.cn/TSmiR) has been developed to provide information on interaction maps of transcription factors and tissue specific miRNAs from experimentally validated and predicted data [Toxoplasma gondii [P. berghei, P. yoelii, P. chabaudi, Cryptosporidium parvum, Mouse hepatitis virus, and Staphylococcus aureus [The existence of tissue-specific miRNA signatures has been suggested in several studies –108, reited data . miRNAs ted data . There ated data , hepatitted data , human ited data , Epsteinted data , bacterited data , 115 as ted data . In filated data  and someted data . Few miRa gondii . Specifis aureus , or in dPlasmodium infection, the current knowledge about the role of miRNAs in malaria disease is limited.Although it is known that miRNAs regulate the immune system  and may Plasmodium species lack the canonical RNAi machinery of other eukaryotes. No dicer or argonaute proteins have been found in any of the ten species of Plasmodium with sequenced genome available, and exhaustive analyses using RNA-based gene silencing approaches have revealed that Plasmodium lacks the enzymology required for RNAi-based ablation of gene expression [Plasmodium is not surprising assuming that RNAi could initially have evolved as a host defence mechanism against viruses and transposons. Plasmodium is not affected by viruses, and some authors consider that the loss of RNAi machinery in some parasites may provide them with an advantage to develop new strategies to escape host defences [Plasmodium, several studies have shown successful down-regulation of gene expression using dsRNA, suggesting the existence of non-canonical RNAi pathway, albeit not found until now [pression . The absdefences , 123. Alntil now –126.Plasmodium, gametocytes of the parasite have to be taken from the blood of infected vertebrate hosts by female Anopheles mosquitoes. Plasmodium parasites then invade the midgut of the mosquito, a critical step for the parasite because the host tries to eliminate the infection. The knockdown of mosquito dicer and argonaute 1 has been shown to lead to increased sensitivity to Plasmodium infection [Plasmodium infection in the mosquito is dependent on the stages along parasite progression [To complete the complex life cycle of nfection , suggestgression , 129.Plasmodium [Toxoplasma to regulate host gene expression [Plasmodium mRNAs to block their translation [Plasmodium and recognize target mRNAs, if it only happens in sickle cells or in all erythrocytes, and whether these miRNAs from the erythrocyte can get into the circulation and attack surrounding infected cells. Other small regulatory RNAs recently found in malaria parasites, such as circular RNAs (circRNAs) [All miRNAs isolated from malaria-infected erythrocytes have been confirmed to be of human origin, suggesting there are no miRNAs of asmodium , 131. Thpression . Howevernslation . In partnslation  and its ircRNAs) , which hircRNAs) , may alsPlasmodium infection in rodent models. Reprogramming of host miRNA expression was observed in the female mouse liver after a primary infection with P. chabaudi. These changes persisted during re-infection in mice that have acquired some level of anti-malarial immunity, suggesting that some miRNAs may regulate the development of protective immunity against malarial blood stages of P. chabaudi [P. berghei infected mice with cerebral and non-cerebral malaria. Alterations of certain miRNAs coincided with increased levels of circulating inflammatory cytokines and apoptosis within the brain [P. chabaudi infection. Although authors suggested that alterations of miRNAs allowed the upregulation of numerous target genes in response to infection, causal relationship between altered miRNA levels and pathology cannot be established [Only four studies have looked for abnormal regulation of host miRNAs after a chabaudi . Neverthhe brain . A thirdablished . Finallyablished . More imPlasmodium infections. A study compared the level of few miRNAs detected by quantitative PCR in the plasma of 19 individuals infected with malaria  with levels of 19 non-infected controls. The decrease of two miRNAs, miR-451 and miR-16, was correlated not only with the infection of Plasmodium but also with its severity, defined on the basis of parasite densities [Plasmodium infection. Moreover, the down-regulation of miR-451, the most abundant miRNA in human erythrocytes, is surprising given that malaria infections usually generate high haemolysis and therefore release miRNAs from erythrocytes. Further studies are needed to explore the value of miRNAs as biomarkers of malaria infection.Very little is known about the potential of plasma miRNAs as markers of ensities . HoweverP. falciparum to host receptors can trigger intracellular signals in target cells [P. falciparum, although not able to produce miRNAs, may manipulate host miRNAs, pointing to the potential of these small molecules to not only shed light on the molecular mechanisms involved in severe malaria and sickle cell resistance [P. falciparum [P. falciparum sequestration, for instance at the brain, lung, spleen or liver, might have the potential to be developed into a biomarker of malaria-specific severe disease (Fig. P. falciparum sequestration may allow discriminating infected individuals with significant parasite sequestration from those in whom the parasitaemia is unrelated to the cause of severity [The discovery of miRNAs has opened exciting possibilities to identify blood biomarkers of organ-specific injury. The content of miRNAs in the host is influenced by host-pathogen interactions, as has been shown for bacteria, viruses and apicomplexan parasites , 142, inet cells  and poteet cells , vasculaet cells . These psistance , but alslciparum . Thus, mseverity . Such anP. vivax or P. ovale [Composite diagnostics, including nucleic acid amplification of the pathogen and miRNA detection, to ascertain the pathological cause of the severe disease could thus guide effective therapy, ensuring that children receive prompt and correct treatment, and reducing the development of resistance. Such miRNAs can be also the basis for new diagnostic tools to predict malaria infections at risk of progression to severe disease that may require a more aggressive treatment. Moreover, miRNAs associated with malaria-related pathologies may also be applied to track the success of treatments against severe malaria cases, to generate estimates of malaria mortality rates, to evaluate outcomes in severe malaria clinical trials, and to improve measurements of the impact of future interventions against severe malaria. Finally, miRNAs could be also informative of the initial stages of liver infection, including hypnozoites in the case of P. ovale , hidden P. ovale , and of P. ovale . FinallyP. falciparum or to make host cells refractory to parasite cyto-adhesion. In addition, this knowledge can provide important clues about the physiopathological mechanisms involved in other infections and help to dissect the biogenesis and function of miRNAs in normal human cells.Robust and affordable point-of-care tests based on miRNA detection with laminar flow-assisted dendritic amplification on power-free microfluidic chip , or futupost-mortem samples can be obtained. This approach opens the exciting possibility of identifying new diagnostic targets for patients at risk of severe malaria, and to distinguish severe malaria cases from those associated with other common pathologies, and also to facilitate treatment decisions. Overall, this knowledge will open up new horizons and opportunities for research, not only on malaria and other infectious diseases, but also on the role of human miRNAs. Future studies will likely contribute to identify key blocking events that may be targeted to develop new effective methods for inhibiting or reversing the process of cyto-adhesion, as well as to develop miRNA-based biomarkers of diagnostic value for severe disease associated with P. falciparum sequestration.Although the enormous potential of miRNAs as a non-invasive biomarker for several human diseases is well known, little has been done to decipher their relationship with malaria pathogenesis. However, evidence is accumulating that host miRNA can be altered by parasite infections and be involved in the course and outcome of infectious diseases , pointin


Plasmodium parasitaemia in households of index cases, and identify risk factors for infection; these data could inform reactive screening approaches and identify target risk groups.As momentum towards malaria elimination grows, strategies are being developed for scale-up in elimination settings. One prominent strategy, reactive case detection (RACD), involves screening and treating individuals living in close proximity to passively detected, or “index” cases. This study aims to use RACD to quantify Plasmodium infection with rapid diagnostic tests (RDT), microscopy and real-time polymerase chain reaction (PCR). Participants were interviewed to identify risk factors. A comparison group of 60 randomly-selected households was also screened, to compare infection levels of RACD and non-RACD households. In order to identify potential risk factors that would inform screening approaches and identify risk groups, multivariate logistic regression models were applied.This study was conducted in the Western Cambodian province of Pailin between May 2013 and March 2014 among 440 households. Index participants/index cases (n = 270) and surrounding households (n = 110) were screened for Plasmodium vivax, two Plasmodium falciparum); seven were afebrile. Seventeen infections were identified through PCR screening of 1596 individuals . In the control group, 25 P. falciparum infections were identified through PCR screening of 237 individuals, and no P. vivax was found. Plasmodium falciparum infection was associated with fever (p = 0.013), being a member of a control household (p ≤ 0.001), having a history of malaria infection (p = 0.041), and sleeping without a mosquito net (p = 0.011). Significant predictors of P. vivax infection, as diagnosed by PCR, were fever  and history of malaria infection (p ≤ 0.001).Nine infections were identified in households of index cases (RACD approach) through RDT screening of 1898 individuals (seven This study found that RACD identified very few secondary infections when targeting index and neighbouring households for screening. The results suggest RACD is not appropriate, where exposure to malaria occurs away from the community, and there is a high level of treatment-seeking from the private sector. Piloting RACD in a range of transmission settings would help to identify the ideal environment for feasible and effective reactive screening methods. Plasmodium falciparum since 2009 [Due to increased international funding, political will, and a new generation of malaria diagnostic and treatment tools, the target to achieve malaria elimination is growing in momentum –3. As ofnce 2009 .Although active case detection (ACD) is considered an essential element of malaria elimination –13 thereUnlike PCD, RACD is also potentially an effective strategy for detecting afebrile malaria infections, which also tend to cluster in low-transmission settings . HoweverPlasmodium parasitaemia in households of index cases detected by PCD in a low malaria transmission setting, and to evaluate potential risk factors for infection that will inform reactive screening approaches and identify high-risk groups.The objectives of the study presented here are to quantify P. falciparum to anti-malarial drugs since the 1960s [P. falciparum resistance to artemisinin-based treatments was first identified [Pailin province is located in the northwest of Cambodia. This area has repeatedly been the epicentre of emerging resistance of he 1960s , 27, andentified . In receentified . The numPailin has six health centres, staffed with a nurse and a midwife who can test, treat, and track malaria cases. In addition, Pailin has also a malaria alert system delivered through a network of village malaria workers (VMWs). Each of the 114 villages has two VMWs who are trained to diagnose malaria infections using rapid diagnostic tests (RDT), and to provide treatment according to national guidelines. VMWs routinely collect blood films and filter paper blood spots and send a short message service (SMS) to alert the public health facility and the national malaria programme when a new malaria case is identified . At the Plasmodium parasitaemia in households of “index” cases. Index cases were defined as those testing positive for malaria through PCD at a health facility or through a VMW. A team comprised of health centre staff or VMWs (based on catchment areas) and Malaria Consortium staff was mobilized to follow up index cases at their homes within 3 days of the case being detected. Within the household of the index case, all other residents were invited to participate in the study. In addition, for every 15th index case identified, the five nearest households to the index case household were also invited to participate. For every 30th index case, the ten nearest households were invited to participate.RACD was implemented between May 2013 and March 2014 in 114 villages in Pailin in order to quantify the reservoir of afebrile Plasmodium infections reported in MIS data during the previous year (“high incidence” control households) and 30 households were selected from villages with five or fewer confirmed infections during the previous year (“low incidence” control households). The number of cases, rather than incidence, was chosen for purposes of stratification due to the unstable nature of village populations and unreliable estimates of village populations in Cambodia. Due to logistic reasons, the control households were not screened throughout the study period; and therefore were all, therefore, screening of all control households was conducted completed at the end of the study period.In addition, 60 randomly-selected control households were also screened. The randomly-selected households were stratified by case numbers; 30 households were selected from villages with more than five confirmed Plasmodium infection, 16 % had other individuals in the same household found to be Plasmodium-infected using microscopy. Therefore, it was estimated that with PCR, a more sensitive diagnostic tool, additional Plasmodium-infected individuals would be identified in 20 % of households with an index case. With a precision of 5, 95 % confidence interval and expected refusal rate of 10 %, it was estimated that 270 index cases should be followed up and included in the study to evaluate if 20 % of index households had at least one afebrile infected individual. The total number of households required with this expected proportion , a precision of 5 % (e), and a 95 % confidence interval (Z = 1.96) was N = (Z2pq)/e2~246 households +10 % refusal rate 270 index cases (households).All villages in Pailin were considered eligible to be included in this study. Preliminary analysis of microscopy data from the national malaria survey conducted in Cambodia in 2010 indicated that among households with at least one P.f/Pan and P.f/P.v), single thick and thin blood film, and three blood spots on filter paper for subsequent analysis by PCR. Index cases did not provide a blood sample at the household; only their original RDT diagnosis from the health centre or VMW was available. In addition, a structured case investigation form was completed by interviewing the head of each household, in order to collect information about demographic indicators and potential risk factors, including travel history, work history, and use of malaria prevention methods by the individual residents in the household. History of malaria infection was defined as having ever had malaria in the past. Net use was defined as having slept under a mosquito net (either standard or treated nets) the night before being interviewed. All responses to questions were self-reported.All members of selected households were invited to participate in the study. Each member was asked to provide a single finger-prick blood sample for preparation of multi-species RDT  up to 3 weeks after collection until they were transferred to the Institut Pasteur in Cambodia (IPC) for PCR analysis. Each blood spot was cut with a sterile 3 mm diameter hole-puncher and placed in a 96-well plate in numerical order. Samples were lysed overnight in a Saponin solution, and DNA was subsequently extracted using InstaGene Matrix resin, as previously described . Samplesmalariae .Paper-based questionnaires were anonymized and double-entered, along with all laboratory results, into an EpiData  template, which included range and type checks to minimise entry errors. PCR results were supplied by IPC in Microsoft Excel format, and merged with the core data using a unique ID code. However, due to variations in quality of slides prepared by local health staff and missing slides, it was decided to exclude all microscopy data from the analysis.versus afebrile cases. Logistic regression models were developed to investigate individual level risk factors associated with RDT and PCR-identified infection. In addition, the proportion of additional individuals positive by PCR and RDT that would have been identified using screening approaches based on different risk factors  was estimated.Data were analysed in STATA software, version 12.0 . Index case demographics and risk factors were compared to non-index cases to better understand what might lead to greater risk of infection. Infection rates and afebrile parasitaemia were compared between different household categories . The proportion of screened household members with infection was also stratified by village incidence  and by febrile Plasmodium positivity by species as the primary outcome for both RDT and PCR diagnostic tests. However, due to the poor fit of the RDT models resulting from low case numbers, both species were combined for a multivariate RDT model.To explore the factors significantly associated with RDT- and PCR-identified infection, univariate logistic regression models were developed along with multivariate logistic regression models. These models used a backward step-wise approach, re-testing all included variables in the final minimal model. Predictors found to be borderline significant p value <0.1 were kept in the multivariate model. Models allowed for within-household correlation. Separate models were prepared with The variables included in the model presence of fever, household category, distance to forest, village burden based on 2013 Malaria Information System data, history of malaria infection, being an adult male, and net use. Presence of fever was defined as having a temperature >37.2 °C using a digital axillary thermometre. Household categories included index households, the five closest households to index cases, and the ten closest households to index cases, as well as high incidence and low incidence control households. Distance to forest was categorized as <500 m, 500 m to 1 km, 1–2 km, and 2–5 km. Village burden was categorized as those greater than or less than five cases in 2013.This study was approved by the Cambodian National Ethics Committee for Health Research (0064 NECHR). Blood safety was ensured by collecting samples using sterile techniques. VMWs were already fully trained in blood collection methods, but additional training was given on specific RACD procedures before the study took place. Signed consent was requested from the household head and individuals or their guardians (in the case of children) before taking blood samples, administering questionnaires, and any other form of data collection. Individuals who had positive RDTs were provided with treatment according to the Cambodian National Treatment Guidelines.P. vivax infection  had  % had P.P. vivax infections and two (22 %) had P. falciparum infections. Seven (78 %) infections were afebrile (Table P. vivax infections (88 %) and two (12 %) being P. falciparum/P. vivax-mixed infections. Of the 16 PCR-positive individuals who had their temperature taken 14 (88 %) were afebrile. The proportion of individuals testing positive in index households (1.3 %), and the five (0.5 %) or 10 (0.9 %) nearest households was similar between all categories . Seven (78 %) had  and two 2 % had PP. falciparum (16 % in the high risk villages and 5 % in the low risk villages). While the majority of infections from households identified through RACD were P. vivax, all PCR positive infections in the control households were P. falciparum. From those who had their temperature taken (21/25) 71 % were afebrile.None of the 183 people screened by RDT in control households were positive. Out of 237 people from control households tested by PCR, 25 (10.5 %) were positive with all infections being P. falciparum positive by PCR were: presence of fever ; living in a randomly selected high incidence control household ; living in a randomly selected low incidence control household ; history of malaria infection ; and sleeping under a mosquito net the night before . Interestingly, sleeping under a net was associated with protective effect despite the usage of nets being almost universal (96 %). The main predictor P. vivax infection by PCR was history of malaria infection , and a borderline statistical significance was found with fever , , and history of malaria infection . Mosquito net use was not included in the model since all RDT positive individuals slept under a net the night before. Risk factors found to be significant predictors for being P. falciparum PCR-detected cases, or 27 % of all infections, and no additional RDT-detected cases. Only 7 % of individuals would have required screening. Adding history of malaria as a criterion would have identified five additional P. vivax infections, one additional P. falciparum infection, and one additional P. falciparum/P. vivax mixed PCR-detected case , as well as two additional RDT-detected infections (44 % of RDT-sensitive infections), with 21.7 % of individuals requiring screening.If fever had been the only criterion for screening in this study, only 22 % of both RDT- and PCR-detected infections in the population would have been identified, with only 4 % of individuals requiring screening. Adding non-use of bed nets to the criterion would have identified two additional Plasmodium parasitaemia in households of index cases detected by PCD in this study was extremely low (0.5 % by RDT and 1.1 % by PCR), suggesting that screening of index households with RDT or PCR may not reveal as many additional cases in this setting as originally hypothesized. There was little difference between the reservoirs identified in index households versus neighbouring households, suggesting that targeting an RACD strategy to index households alone is insufficient to identify all infections in this setting. This is inconsistent with other studies using similar RACD approaches; nevertheless in those studies the difference in positivity between the household categories was small (1–2 %) and the number of cases quite low (23–74) [ (23–74) , 13.Plasmodium parasitaemia. In fact, many RACD strategies in other settings already use fever as a screening tool [Fever was found to be a main risk factor associated with ing tool . Absenceing tool . This suP. falciparum infections, while the majority of index and surrounding household’s infections were P. vivax infections. This result was not consistent with another RACD study in Zambia, where much higher infection rates were found in index (8 %) than control households (1 %) [P. falciparum outbreak, which may have started after completion of other RACD screening. However, government surveillance data does not show any P. falciparum cases in the months before, during, or after control households were sampled . Data from the 2013 Cambodia Malaria Survey found that although 28.7 % individuals with fever sought treatment at public facilities (including VMWs), 58.3 % went to private providers, and 12.9 % did not seek treatment [It is important to note, however, that all control household infections were ds (1 %) . Controlreatment . It is pPlasmodium parasites, which would allow us to determine if infections within the index households were similar to, and thus likely transmitted by, the index case. This study also relied on VMWs or health facility staff to collect blood samples from households; consequently the blood slides were of varying quality. Similar limitations may be faced when implementing RACD in other settings and special efforts to improve training and supervision should be taken to ensure high quality of slides.During the study it was not possible to collect any samples for PCR analysis from index cases. As a result, it was not possible to conduct molecular analysis and genotyping of identified Recent research suggests that sub-microscopic infections may not substantially contribute to the infectious reservoir . These lAnother limiting factor for RACD is the location of exposure. If the majority of exposure to infection occurs outside the community, then screening people according to residence is not appropriate. The results of this study strongly suggest that there is limited local transmission around the household, since very few secondary infections were identified. Therefore in this setting, RACD does not effectively target those most likely to have afebrile parasitaemia; however it may be more appropriate in settings with slightly higher transmission and different vector species. In order to better determine the incidence threshold at which RACD successfully identifies secondary infections, it is recommended that similar studies be conducted across a range of transmission settings.In addition to questions about the effectiveness of RACD’s effectiveness for that specific setting, logistical feasibility must also be considered. For example, it was found that a team of 5–6 people employing this approach could visit two households per day and screen up to six household members. The time it takes to reach households may be significantly increased if RACD is adopted on a larger scale and integrated into the national programme’s routine activities, as was shown in Swaziland . The levRecent research shows that Mass Screening and Treatment (MSAT) may be more effective than individual screen and treat strategies, suggesting MSAT may be needed to successfully treat the afebrile parasite reservoir and reduce transmission , 25, 35.In most settings, introducing a new case investigation and response methodology requires a fundamental shift in the way surveillance is carried out at peripheral level. Given the limited evidence available, number of different options available, and due to the varying epidemiology between countries, it is unlikely that there will be a “one size fits all” solution. If elimination programmes find that RACD integrates well with their strategic plan, a careful evaluation of necessary methods, targeting, population coverage, interventions, and impact is in order .There may also be a need to combine different approaches, or apply different strategies in different transmission settings within the country. Researchers are also working to improve data that can be used to drive these approaches. One example is the use of high spatial and temporal resolution surveillance data to identify hotspots , 39. ComAll PCR analyses conducted were subject to quality assurance procedures by qualified and experienced laboratory technicians, including positive and negative controls for both the extraction and PCR stages, and any invalid run was repeated in full. It has been shown that PCR analysis of small blood volumes (e.g. 5 µL) eluted from dried blood spots may miss some low density infections which are identifiable with PCR on larger volume venous blood samples (1 mL) , howeverThis study found that RACD identified very few secondary infections when targeting screening to index and neighbouring households within a community. It is recommended that RACD is not appropriate for very low transmission settings where exposure to malaria occurs away from the community (e.g. in forested areas), and there is a high level of treatment-seeking from the private sector. RACD could be adapted to include index cases from the private sector, but is most appropriate in settings with declining and heterogeneous transmission, settled and stable populations, and where the primary vector habitat is within communities.


More human death and disease is caused by malaria parasites than by all other eukaryotic pathogens combined. As early as the sequencing of the first human genome, malaria parasite genomics was prioritized to fuel the discovery of vaccine candidate antigens. This stimulated increased research on malaria, generating new understanding of the cellular and molecular mechanisms of infection and immunity. This review of recent developments illustrates how new approaches in parasite genomics, and increasingly large amounts of data from population studies, are helping to identify antigens that are promising lead targets. Although these results have been encouraging, effective discovery and characterization need to be coupled with more innovation and funding to translate findings into newly designed vaccine products for clinical trials. Plasmodium parasites infecting humans via transmission by Anopheles mosquitoes Plasmodium falciparum is most prevalent. Existing interventions need to be more widely applied, including insecticide-treated bed nets to reduce mosquito biting, antimalarial drug prophylaxis targeted to vulnerable groups, and access to prompt diagnosis and treatment. An effective vaccine could be a cost-effective additional means of controlling malaria The drive to sequence malaria parasite genomes began in the 1990s with a bold vision for ‘vaccinomics’ which would showcase the benefits of genomics to society A key issue for malaria vaccine design is selecting the most appropriate parasite life-cycle stage to be targeted  . AP. falciparum containing approximately 5500 genes encoded on 14 chromosomes with a complete haploid genome size of ∼23 Mb P. vivax, the second most important cause of human malaria P. knowlesiP. falciparumThe first genome sequence of a malaria parasite was published in 2002 for a cultured strain of A degree of immunity to malaria is generally acquired by experience of the infection, as shown by experimental studies in the first half of the 20th century on patients with neurosyphilis. Fevers induced by administering malaria parasite infections were beneficial to such patients in the age before antibiotics, because elevated temperatures killed spirochetes more effectively than other available treatments, but many patients became immune to repeated malaria infections Parasites may be attenuated at the infective sporozoite stage which invades hepatocytes P. falciparum CSP  in erythrocyte invasion was identified by mapping this locus for ability to invade Aotus monkey erythrocytes It is notable that some important targets of immunity are encoded by genes occurring only in eveloped . HoweverP. falciparum has also undergone scale-up Parasite genetic cross experiments map loci down to a few cM, typically in the order of approximately 50 kB which may contain upwards of 10 genes, after which targeted gene knockout and allelic replacement may identify causal genes. Large libraries of gene knockouts are now available for rodent malaria parasites that are most amenable to genetic manipulation Rh5 gene in P. falciparum was not achieved despite repeated attempts, suggesting that it may be essential to parasite growth at the cultured asexual blood stage Rh5 transcript at the merozoite-containing schizont stage appears to vary among parasite isolates from clinical cases Aotus monkey erythrocytes have occurred in a parasite line adapted to these hosts Experimental knockout of the P. falciparum genes at particular life-cycle stages has been described by transcriptome profiling, with particular detail for the cultured 48 h cycle of the asexual blood stages P. falciparum estimated that approximately 10% of all genes exhibit clonally variant transcription Expression of var gene family with approximately 60 copies in each haploid P. falciparum genome encoding variants of the parasite-infected erythrocyte membrane protein 1 (PfEMP1) var gene types in each parasite genome var gene specialization is that the expression of particular genes is associated with severe clinical forms of malaria in children Malaria parasite genes in subtelomeric chromosomal regions show extreme patterns of sequence diversity and clonal expression variation, particularly the P. falciparum termed rifin and stevor encode proteins that are variably expressed on infected erythrocytes and merozoites, although whether antibodies to these are protective is not as well established as for PfEMP1 msp3-like genes clustered in a non-subtelomeric position of the genome Other large subtelomeric gene families in eba, clag/RhopH1, surfin, and msp3-like gene families, supporting the idea that this extracellular stage is particularly susceptible to acquired immunity Pathogen antigens are likely to be subject to frequency-dependent selection from acquired immune responses, and particular patterns of allelic polymorphism may reveal departures from neutral expectations as a consequence. Several antigens already considered to be malaria vaccine candidates first showed such evidence P. vivax strains published to date indicate that this species is more polymorphic than P. falciparum, with nucleotide diversity being approximately twice as high, suggesting that this parasite has been widespread in humans for longer P. vivax candidate antigen gene sequences in population surveys implicate ama1, trap, and dbp as being under balancing selection P. falciparum homologs P. falciparum polymorphisms among a small number of laboratory lines compared with fixed differences between P. falciparum and the chimpanzee parasite P. reichenowiGenome sequences for the small number of de novo assembly may soon capture the substantial polymorphism that exists in these regions Genome-wide scans for loci under selection have not yet incorporated detailed analyses of repeat sequences, subtelomeric chromosome regions, or other loci with highly divergent alleles. Such sequences are not reliably characterized by short-read mapping to reference genomes and are therefore omitted from most analyses. However, advances in longer-read sequencing and methods of P. falciparum AMA1, an N-terminal part of MSP1, and P. vivax DBP The predictive power of analyses to identify patterns of selection on antigen genes should be evaluated by epidemiological and experimental validation of the putative targets. In epidemiological studies, large cohorts of individuals need to be followed in order for levels of naturally-acquired antibodies against an array of different recombinant proteins to be tested for correlations with reduced prospective risk of clinical malaria P. falciparum as a vaccine component P. vivax DBP, such dissection to remove polymorphic sites may not be possible, although particular formulations may still induce strong antibody responses to conserved epitopes Design of malaria vaccines should incorporate discoveries from the above areas of research, although there remains no standardized means or structure for capturing these. Different alternative approaches will remain potentially viable. In particular, vaccines designed to prime immune responses in infants to specific targets of naturally acquired immunity need to either focus immune responses on conserved parts of the antigens or to elicit polyvalent responses that cover the natural antigenic diversity. If polymorphisms are clustered in regions of a primary sequence, it may be possible to use a relatively conserved part of the sequence, an approach that has been followed to focus on a C-terminal sequence of MSP3 of Antigenic diversity in relation to vaccination has been intensively studied for AMA1, in which the particular importance of a highly polymorphic amino acid position has been shown in several studies, including a clinical trial demonstrating allele-specific protection There remains no consensus template for an effective malaria vaccine, but the success of multivalent vaccines against viral and bacterial infections supports the rationale to characterize individual targets of immunity in detail to assist the design of subunit vaccines. Engineering of more effective immunogens is vital, and should involve modeled protein design to focus responses on important epitopes


Malaria in pregnancy remains a public health problem in Nigeria. It causes maternal anaemia and adversely affects birth outcome leading to low birth weight, abortions and still births. Nigeria has made great strides in addressing the prevention and control of malaria in pregnancy. However, recent demographic survey shows wide disparities in malaria control activities across the geopolitical zones. This situation has been compounded by the political unrest and population displacement especially in the Northeastern zone leaving a significant proportion of pregnant women at risk of diseases, including malaria. The use of malaria preventive measures during pregnancy and the risk of malaria parasitaemia, anaemia and low birth weight babies were assessed among parturient women in an insurgent area.A cross-sectional survey was conducted among 184 parturient women at Federal Medical Centre, Nguru in Yobe state, between July and November 2014. Information on demographics, antenatal care and prevention practices was collected using an interviewer-administered questionnaire. Maternal peripheral and the cord blood samples were screened for malaria parasitaemia by microscopy of Giemsa-stained blood films. The presence of anaemia was also determined by microhaemocrit method using the peripheral blood samples. Data was analysed using descriptive and analytical statistics.Prevalence of malaria parasitaemia, anaemia and low birth weight babies was 40.0, 41.0 and 37.0 %, respectively, and mothers aged younger than 25 years were mostly affected. Eighty (43.0 %) of the women received up to two doses of sulfadoxine-pyrimethamine for intermittent preventive treatment (IPTp-SP) during pregnancy and most, 63 (83.0 %) of those tested malaria positive received less than these. Presence of malaria infection at antenatal clinic enrollment , non-adherence to direct observation therapy for administration of IPTp-SP  and receiving <two doses of IPTp-SP  were significant risk factors for malaria parasitaemia at delivery.The high prevalence of malaria in pregnancy and the adverse outcome in this insurgence area reflects the poor access of pregnant women to preventive measures such as IPTp-SP. Effort to reach displaced pregnant women and supervision of delivery of malaria preventive measures by healthcare providers should be intensified. In Nigeria, malaria remains a public health problem and accounts for 11 % of maternal mortality . The 201This study was conducted from June to November 2014 at Federal Medical Center, a tertiary and referral health institution in Nguru, Yobe State, Nigeria. It is a 390-bed hospital and has an obstetrics and gynaecology department that provides antenatal care services. The antenatal clinic (ANC) runs 2 days per week (both for bookings and check-up), and the services rendered include health education, provision of IPTp-SP, distribution of ITNs, routine laboratory and radiological investigations. Nguru town is a malaria endemic area with transmission all the year round and peaks during the rainy season [Descriptive cross-sectional study design was used. The study population was parturient women delivering at FMC, Yobe. They must have been booked, had attended antenatal clinic in FMC, Nguru and presented in labour at term (between 36 and 40 weeks of gestation). Those with an eventful antenatal period, coexisting premorbid condition, preterm labour and who had no antenatal care and supporting records were excluded.The study minimum sample size of 168 was calculated using sample size formula for single proportion with 12.5 % prevalence of malaria parasitaemia at delivery from a study in Sokoto , precisiStudy participants were recruited consecutively as they presented during labour until the sample size was attained. Only those who had available supporting ANC record were included in the study. Written informed consent was obtained from each study participant or guardians for mothers less than 18 years of age before enrollment.Trained laboratory scientists collected 5 mls of peripheral blood from the mother using vacutainer cup and needle and cord blood from the newborn into separate EDTA bottles from each study participant. Each sample was given a number and paired (mother-newborn) and labeled with the patient’s information. Thick and thin blood films were made from both venous and cord blood, then stained with Giemsa. They were examined microscopically using oil immersion objective (×100) for the presence of malaria parasite. Parasites were identified and densities estimated by counting against 200 leucocytes , 7.Trained research assistants administered a structured pre-tested questionnaire to collect information on demographics, malaria preventive strategies used, doses and trimester of administration of IPTp-SP and haematocrit values during the ANC visits and at delivery. Data on previous use of IPT, malaria infection and treatment during pregnancy were extracted from the ANC records. Digital weighing scale for babies was used in the labour ward by skilled midwives to measure the baby’s weight and recorded in the labour ward delivery register. Baby’s birth weight was collected from labour ward record. Low birth weight (LBW) babies were categorized as those with birth weight <2500 g .Data collected for the study were entered, cleaned and analysed using Epi-info version 3.7 and Microsoft excel. Means, standard deviation and proportions were computed as relevant to summarize the data. Bivariate analysis (Chi square test) was used to determine association between categorical. Multiple logistic regression was carried out to determine predictors of malaria parasitaemia. Level of significance was set at 5 %.The ethical approval for the conduct of the study was obtained from the Institutional Ethics Review Committee of Federal Medical Center, Nguru. Written informed consent was obtained from each study participant or from guardians for mothers less than 18 years of age whose assents were also sought. Results of those found to have anaemia and parasitaemia were provided to the obstetricians for appropriate management of the patients. Information collected from the participants was kept confidential and stored in both hard-locked in cabinets and password- protected electronic files. Non-personal identifiers were used during analysis and presentation.Overall, there were 268 women delivered at the health facility within the study period and 184 pregnant women in labour who met the inclusion criteria, were recruited and interviewed. The mean age of the respondents was 25.1 ± 2.5 years. More than half of them (54.0 %) were aged between 15 and 24 years, and the majority were primi- and secundigravidae (62.0 %).Of the 184 parturient women, 74 (40.0 %) were positive for malaria parasites, 76 (41.0 %) had anaemia and 68 (37.0 %) had LBW babies (Table Only 80 (43.0 %) of the parturient women received up to two or more doses of IPTp-SP, while 57.0 % received only a dose or none. Majority of the women who had taken up to two doses were primi- and secundigravidae 45 (40.0 %), while only 35.0 % of the multigravidae had taken up to two doses. Almost all the respondents 173 (94 %) mentioned they slept under ITNs in the night preceding the interview.Of 30 primi- and secundigravidae who delivered LBW babies, 19 (63 %) and 6 20 %) had one and at least two doses of IPTp-SP respectively. Of 38 multigravidae who delivered LBW babies, 32 (84 %) and 4 (11 %) had one and at least two doses of IPTp-SP, respectively  used during pregnancy  presence of malarial infection at ANC enrollment  and method of IPT administration by prescription  were significantly associated with presence of malaria parasitaemia among the parturient women , presence of malaria parasitaemia at ANC enrollment and not have IPT administered as DOT method. Similar studies that logically linked the presence of parasitaemia to various parameters were done in Benin , 15 wherLimitations of this study include the reliance on patients recall with regards to the administration of IPT through prescription (not DOT) and use of LLINs. Moreover, evidence of prescription from the patients records provides confirmation of IPT utilization, unlike those considered as LLIN users based on report of having slept under a LLIN the night before the study.The effective use of malaria preventive strategy (IPTp-SP) was generally low and there was high prevalence of malaria parasitaemia, maternal anaemia and low weight babies. It is recommended that providing adequate information to pregnant women by health care providers can empower them with knowledge to demand for DOT mode of IPTp-SP. Also establishing a reliable mechanism by Government for ensuring adherence to DOT-based administration of appropriate dose for IPTp-SP is crucial.