New regimens for intermittent preventive treatment in pregnancy (IPTp) against malaria are needed as the effectiveness of the standard two-dose sulfadoxine-pyrimethamine (SP) regimen is under threat. Previous trials have shown that IPTp with monthly SP benefits HIV-positive primi- and secundigravidae, but there is no conclusive evidence of the possible benefits of this regimen to HIV-negative women, or to a population comprising of both HIV-positive and –negative women of different gravidities.Plasmodium falciparum malaria at delivery diagnosed with a real-time polymerase chain reaction (PCR) assay.This study analyzed 484 samples collected at delivery as part of a randomized, partially placebo controlled clinical trial, conducted in rural Malawi between 2003 and 2007. The study included pregnant women regardless of their gravidity or HIV-infection status. The participants received SP twice (controls), monthly SP, or monthly SP and two doses of azithromycin (AZI-SP). The main outcome was the prevalence of peripheral P. falciparum malaria at delivery was 10.5%. Compared with the controls, participants in the monthly SP group had a risk ratio (95% CI) of 0.33  and those in the AZI-SP group 0.23  for malaria at delivery. When only HIV-negative participants were analyzed, the corresponding figures were 0.26  for women in the monthly SP group, and 0.24  for those in the AZI-SP group.Overall prevalence of PCR-diagnosed peripheral Our results suggest that increasing the frequency of SP administration during pregnancy improves the efficacy against malaria at delivery among HIV-negative women, as well as a population consisting of both HIV-positive and –negative pregnant women of all gravidities, in a setting of relatively low but holoendemic malaria transmission, frequent use of bed nets and high SP resistance.ClinicalTrials.gov NCT00131235 Plasmodium falciparum infections every year P. falciparum in SSA Malaria is one of the most important preventable causes of poor maternal health and adverse birth outcomes At least four trials have tested the efficacy of an IPTp regimen that contains monthly SP-dosing In our previously reported trial from Malawi we included pregnant women regardless of their gravidity or HIV-infection status P. falciparum parasitaemia than earlier diagnosed with microscopy In a subsequent study, we applied a polymerase chain reaction (PCR) – based method to diagnose malaria from peripheral blood samples collected at delivery in the above described trial. With this more sensitive method, we documented an almost five fold higher prevalence of P. falciparum as part of the Lungwena Antenatal Intervention Study (LAIS), which has been described in detail elsewhere This study is a secondary analysis of peripheral blood samples collected at delivery for real-time PCR assay targeting http://www.clinicaltrials.gov, trial identification NCT00131235). Only participants who signed or thumb-printed an informed consent form were enrolled in the study.The trial was performed according to Good Clinical Practice guidelines and the ethical standards of Helsinki Declaration. The protocol was approved by the College of Medicine Research and Ethics Committee, University of Malawi, Malawi and the Ethical Committee of Pirkanmaa Hospital District, Finland. Key details of the protocol were published at the clinical trial registry of the National Library of Medicine, Bethesda, Md, USA  received otherwise the same treatment, but SP was given monthly from enrollment until 37 gestational weeks. Participants in the second intervention group (“AZI-SP”) received otherwise the same treatment as the monthly SP group, but instead of placebo, they received active azithromycin twice : at enrollment visit and between 28th and 34th weeks of gestation. All participants received ferrous sulphate (200 mg/day) and folic acid (0.25 mg/day) throughout pregnancy.Participants in the control group (“control”) received standard Malawian antenatal care including IPTp with SP , and a placebo to azithromycin, both given twice during pregnancy: at enrollment and between 28th week visit received the normal pre-packed study drugs which included SP for all study groups. Laboratory-confirmed malaria at any other point was treated with quinine .Participants diagnosed with malaria at enrollment or at 28–34SP tablets were purchased from Malawi Central Medical Stores that were supplied by Pharmanova, Ipca Laboratories Ltd, F.Hoffmann-La Roche Ltd, and Universal Corporation Kenya Ltd. Both active azithromycin and its placebo were manufactured and donated by Pfizer Inc. We did not perform any pharmacological tests on the study drugs.A researcher not involved in data collection generated a randomization code list in blocks of nine. Based on this list, an individual drug box was pre-packed for each identification number. The drug box contained appropriate study drugs for each planned study visit in separate opaque drug envelopes labeled with identification number and visit information. Individual slips containing unique identification numbers, but not group allocation, were sealed in individual opaque randomization envelopes. The envelopes were grouped into randomization blocks to ensure similar allocation rates to each group.At enrollment visit, the research personnel interviewed individuals interested in participating in the study about their socioeconomic status, obstetric history and bed net use, gave pre-test HIV-counseling, and performed an antenatal and laboratory examinations. Testing for HIV was optional. Eligible individuals signed or thumb-printed informed consent and picked one randomization envelope which contained an identification number. A research assistant not involved in outcome assessment gave the corresponding pre-packed study drugs to the participant under direct observation.At follow-up visits  the research personnel conducted an antenatal examination. The participants were offered post-test HIV counseling and prevention of mother to child transmission. At the visit during 28th–34th weeks of gestation, a malaria test was conducted. At each visit, the participant took the appropriate pre-packed study drugs under direct observation.Upon notification of a delivery, a research assistant visited the delivery site. She collected data on delivery, examined the newborn and gave her/him nevirapine or placebo based on maternal HIV status. From participants who delivered at a local health facility, maternal peripheral venous blood was collected as thin and thick blood films, and as dried blood spots on filter paper.P. falciparum parasitaemia at delivery was diagnosed with a P. falciparum lactate dehydrogenase-specific (pfldh) real-time PCR assay using DNA extracted from dried blood spots, as previously described Peripheral blood Statistical analysis was carried out with Stata 9.2 . We estimated risk ratio and risk difference for comparison of binary end-points at a single time point. To prevent inflated type I errors due to multiple comparisons, we began hypothesis testing with global null hypotheses of all three groups being identical before doing pair-wise comparisons. We tested the hypotheses with Fisher’s exact test.The proportion of women with no previous pregnancies was higher in the control and monthly SP groups than in the AZI-SP group, and the proportion with peripheral malaria parasitaemia at enrollment was higher in the control group than in the intervention groups. As sensitivity analyses, we adjusted for these two covariates as categorical variables by generalized linear models and compared the adjusted and main analyses. We performed tests for interaction between the interventions and HIV status (those whose status was known), the number of previous pregnancies , and bed net use using the likelihood ratio test, and did analyses stratified by the same variables.P. falciparum, and they formed the study population  number of scheduled SP treatments received was 2.0 (0.2) in the control, 4.2 (0.9) in the monthly SP, and 4.1 (0.8) in the AZI-SP group. Women in the AZI-SP group received a mean (SD) of 2.0 (0.1) azithromycin doses. The mean number (SD) of quinine doses given at non-scheduled outpatient visits was 0.04 (0.23) for the control, 0.05 (0.22) for the monthly SP, and 0.02 (0.15) for the AZI-SP group (P = 0.415). Against the trial protocol some SP-doses were given also at the non-scheduled visits, but the number of these was very small and there were no differences between the groups .P. falciparum malaria at delivery was 10.5%, with significant differences between women in the control group and those who received monthly SP either alone or in combination with azithromycin  of 0.26  and those in the AZI-SP group 0.24  for PCR-diagnosed malaria at delivery, when compared with the controls . MalariaP. falciparum malaria parasitaemia at delivery, respectively. In stratified analyses, HIV-negative participants of all gravidities, primigravidae regardless of their HIV-status, and women who did not use a bed net at enrollment had significantly less malaria in both intervention groups than those in the control group.The use of IPT during pregnancy can prevent malaria and the adverse outcomes it causes to pregnant women and their fetuses in SSA. In the current analysis we used a sensitive PCR–based diagnostic method to compare the antimalarial effect of the standard two-dose SP IPTp regimen with monthly SP, alone or in combination with two doses of azithromycin. Compared with the control group, the participants in the monthly SP and AZI-SP groups had relatively 67% and 77% lower prevalence of PCR-diagnosed peripheral P. falciparum malaria parasitaemia at delivery in the target population from which the sample was drawn.The study population was drawn from a trial which used broad inclusion criteria, random group allocation and blinding of the outcome assessors A secondary analysis of pooled data from three other clinical trials from Kenya, Malawi and Zambia suggested that compared with the two-dose SP regimen, the monthly regimen had a beneficial effect on malaria prevalence at delivery and birth weight among HIV-positive pregnant women in their first or second pregnancy P. falciparum malaria parasitaemia at delivery in the latter, wider target group, as well as among HIV-negative women regardless of their gravidity. This finding is consistent with the results of a recently reported trial from Mali, which found that IPTp with three doses of SP is superior to two doses in reducing malaria prevalence at delivery, LBW and premature delivery among mainly HIV-negative women of different gravidities Our results indicate that IPTp with monthly SP can in some conditions reduce the prevalence of peripheral blood Besides the apparently improved efficacy against malaria, monthly dosing would solve some of the problems associated with the two-dose SP IPTp. There are concerns that the dosing interval in the two-dose regimen might be too long even in areas with low levels of SP resistance, and increasing resistance further shortens the duration of the post-treatment prophylactic effect P. falciparum associated with resistance to SP has increased in Africa, and is very high among pregnant women in some parts of Malawi However, before adopting a monthly SP IPTp policy, several issues have to be taken into consideration. The prevalence of mutations in In our previous analysis we found that the combination regimen of monthly SP and two doses of azithromycin, but not monthly SP alone, was significantly superior to the standard regimen of two SP doses in preventing preterm delivery and LBW P. falciparum malaria In the current analysis, we found that the monthly SP regimen was associated with statistically significant reductions in PCR-diagnosed malaria prevalence at delivery both alone, as well as in combination with azithromycin, and that there were no major differences between the two intervention regimens in the point-estimates for the effect size. The results thus suggest that the decrease in parasitaemia at delivery was mainly caused by the increased frequency of the SP dosage, while the improvement in birth outcomes might have been due to some other activity of azithromycin, independent of its activity against malaria. A possible explanation for azithromycin’s weak antimalarial effect in our study is the used dosage. Although 1g dose of azithromycin given twice several weeks apart is sufficient to cure many RTIs, it might be suboptimal against Two limitations of our study are that we analyzed the prevalence of malaria parasitaemia with PCR only at delivery, and only from peripheral blood. It could be argued that the difference in parasitaemia at delivery simply reflects the fact that the last dose in the regimens containing monthly SP was given nearer to delivery than in the two-dose regimen. Thus we cannot rule out that the groups might have had comparable malaria prevalence during pregnancy and that the differences only developed towards delivery. However, it is unlikely that the efficacy of SP would have differed during pregnancy, and therefore the monthly dosing with shorter treatment intervals is likely to have been more effective throughout pregnancy. This is supported by our finding of a comparable effect size in the prevalence of microscopic malaria parasitaemia at 32 gestational weeks in the original sample from which the current study population was drawn In conclusion, our results suggest that increasing the frequency of SP administration during pregnancy improves efficacy against malaria at delivery among HIV-negative women as well as a population consisting of both HIV-positive and –negative pregnant women of all gravidities in a setting of relatively low but holoendemic malaria transmission, frequent use of bed nets and high SP resistance. However, further research is needed to study the consequences of the prevention of submicroscopic malaria infections to maternal and child health, as well as the effect of different levels of resistance and malaria prevalence on the IPTp efficacy. We therefore recommend the use of sensitive diagnostic tools for malaria, such as PCR and placental histology, and the determination of the level and change of drug resistance and acquired immunity in future IPTp trials.Checklist S1CONSORT Checklist(DOC)Click here for additional data file.Protocol S1Trial Protocol(DOC)Click here for additional data file.


Plasmodium falciparum remains a major cause of death in sub-Saharan Africa. Immunity against symptoms of malaria requires repeated exposure, suggesting either that the parasite is poorly immunogenic or that the development of effective immune responses to malaria may be impaired.Malaria caused by P. falciparum malaria transmission is low and sporadic. Circulating antibodies and memory B cells (MBC) to four malarial antigens were measured using ELISA and cultured B cell ELISpot.We carried out two age-stratified cross-sectional surveys of anti-malarial humoral immune responses in a Gambian village where The proportion of individuals with malaria-specific MBC and antibodies, and the average number of antigens recognised by each individual, increased with age but the magnitude of these responses did not. Malaria-specific antibody levels did not correlate with either the prevalence or median number of MBC, indicating that these two assays are measuring different aspects of the humoral immune response. Among those with immunological evidence of malaria exposure , the median number of malaria-specific MBC was similar to median numbers of diphtheria-specific MBC, suggesting that the circulating memory cell pool for malaria antigens is of similar size to that for other antigens. The immune system's ability to mount an accelerated humoral immune response upon repeated encounter of the same pathogen allows for rapid reduction in disease severity or even complete sterile immunity The immune control of malaria infection is multi-factorial, and there is growing consensus that the synergistic action of antibodies (Ab) and cell mediated effector mechanisms is required for both anti-parasitic as well as clinical immunity Longitudinal studies showing that titres of many anti-malarial Ab decline rapidly in children once parasitaemia is cleared after acute infection In this study, in an area in The Gambia where malaria transmission has declined substantially over the last decade P.falciparum PCR, one individual in each of the 1–4, 15–24 and 25–39 year age groups and three individuals in the 10–14 year age group carried parasites, none of which were detected by slide microscopy.118 healthy volunteers from Brefet, Foni District, The Gambia, were selected using age-stratified randomisation into six different age categories . Venous Giardia lamblia cysts  and one (2.2%) had hookworm ova (13 years old).Stool analysis to assess carriage of intestinal helminths and pathogenic gut parasites was carried out on 46 samples, evenly spread across the different age groups. Two (4.3%) had Morbidity surveillance was performed during the transmission season and study participants suspecting they had malaria, or experiencing symptoms compatible with malaria were asked to report to the village health worker who performed a rapid diagnostic test (RDT). Out of eight participants presenting with symptoms, only one had a positive RDT.At the end of the transmission season, 8 individuals had shown evidence of exposure based on a≥1.5 fold increase to at least one of the malaria specific Ab tested for (see methods section); one of those individuals presented with clinical symptoms compatible with malaria and tested positive by RDT.19, MSP-2, MSP-3, AMA-1 and diphtheria toxoid (DT), and the percentages of individuals having Ab to the different Ag were plotted for each age group  kappa values ranging from 0.55 (MSP-2), 0.64 (MSP-119), 0.72 (DT), 0.78 (AMA-1), to 0.81 (MSP-3).When the data were expressed as MBC/PBMC, the proportion of individuals with malaria specific MBC above the cut-off (hereafter referred to as responders) increased significantly with age for all Ags . RespondTo assess how the breadth of the immune response to malaria Ags develops with age, the average number of malaria Ags recognised by each individual's plasma or cells was stratified by age. As shown in IgG measured in plasma by ELISA is an indicator of the presence of antibody-secreting effector cells (plasma cells) in peripheral tissues or bone marrow whereas ASC detected by cultured ELISpot assay indicate the presence of circulating MBC. To explore the relationship between these two very different measures of the humoral immune response, IgG concentrations were plotted against MBC numbers for all subjects for each Ag  and a coTo explore this data further, we classified each individual's responses to all 4 malaria Ags into the following categories: MBC+ IgG+ , MBC+ IgG− (ELISpot response but no Ab), MBC− IgG+ (no ELISpot response but Ab) or MBC− IgG− (no response in either of the tests). The frequency with which each category occurred in each age group was then expressed as a proportion and is plotted in The proportion of responses to malaria Ags categorized as MBC+ IgG+ increases with age, while the proportion of responses classified as MBC− IgG− decreases , which presumably reflects cumulative exposure to malaria over time. Interestingly however, the proportion of responses classified as MBC− IgG+ also increased with increasing age (Chi squared test for trend: <0.0001) suggesting that antibody secreting plasma cells persist even though the frequency of circulating MBC remains below the threshold of detection.Similar analysis was performed for responses to DT . Since aThe magnitude of MBC responses expressed as % MBC were only poorly correlated with numbers of MBC/PBMC , reflectWe were interested to explore i) whether the magnitude of the malaria-specific humoral and B cell ELISpot responses increases with age, and ii) whether the magnitude of malaria specific B cell ELISpot responses differs from DT specific responses. This requires defining each participant's prior exposure to the antigens. Based on the 100% prevalence of IgG against diphtheria in the study group, it can be assumed that every participant has encountered diphtheria antigen in the past. Determining prior exposure to malaria is less straightforward, especially in this community where the very low levels of transmission over the last 10 years mean that the younger individuals may have had very few (if any) previous malaria infections. Extensive polymorphism of malarial blood stage antigens means that even among exposed individuals, prior exposure to the precise antigenic variants used in our assays cannot be assumed. Also, different individuals may respond differently to a given antigen 19  did not vary significantly across age groups . With th19 , for whi19 .Of note, the magnitude of malarial Ag -specific MBC responses amongst those with evidence of previous malaria exposure was not different from that for diphtheria, irrespective of the chosen denominator . In addition, we repeated the serological survey in December 2009 and tested these samples together with those collected in May 2009. Taking the study population as a whole we observed small, but significant, decreases in the median concentrations of Abs to AMA-1, MSP-1nd MSP-3 , confirmP. falciparum DNA in only one of these 8 subjects. A “rising titre” of serum Ab is widely used as a diagnostic tool and suggests that these 8 individuals were indeed infected with malaria. However, despite the fact that (apart from the one RDT positive case) none of them presented with any signs of illness during the study period, we cannot entirely rule out that the increased malaria Ab titres may be the result of bystander activation of malaria-specific B cells by another asymptomatic infection.However, 8 (including the 1 RDT positive case) out of 99 participants that were tested showed a≥1.5 fold increase in plasma IgG levels to at least one malaria Ag suggesting that they may have recently experienced a malaria infection; 7 out of 8 of these infections are assumed to have been asymptomatic. PCR carried out at the end of the transmission period detected Nevertheless, when we compared the change in frequency  of malaria-specific MBCs among the 8 subjects who had evidence of a recent malaria infection (cases) with the change in MBC frequency in 8 age-matched controls with no evidence of recent infection, the post/pre-season ratio for malaria-specific MBCs  tended to be higher in cases than in controls . While t+ CD27− CD21− and CD10−) have also been reported to be more prevalent in adults from an area with high malaria transmission  than in malaria-naïve volunteers (from the USA), and their proportion was higher among asymptomatic parasite carriers than among non-parasitized individuals + CD27− CD21− and CD10−; + CD27+ CD21+) amongst all B cells was twice as high in children aged more than 5 years than in children aged less than 5 years  in HIV-infected patients compared to healthy individuals  5 years , consistP.falciparum and P.vivax in infrequently exposed Thai adults P. falciparum-specific MBC compartment expands in a step-wise fashion with increasing age and/or repeated malaria infections 19 specific IgG in children <10 years in December 2009 The advent of methods to enumerate circulating MBC Rather surprisingly, given the huge differences in malaria endemicity between the two sites, the age-specific prevalence of both Ab and MBC in The Gambia was remarkably similar to the prevalence reported in Mali A caveat to these interpretations, however, is that the similarity of MBC responses in the different studies may reflect some limitation of the assay system rather than a true biological phenomenon. For example, we find for all Ags, including diphtheria, that although the prevalence of responses varies, the magnitude of the MBC responses (in those classified as exposed) is similar in all age groups. One potential explanation that deserves investigation is that, even in individuals with long-lived humoral immune memory, the maximum precursor frequency of circulating MBCs for any individual Ag is in the order of 1 MBC per million PBMCs and that the spot frequency of 5–10 spots per million PBMC simply reflects the differentiation of these very few MBCs during the 6 days of mitogenic stimulation into MBC. In other words, it may be necessary to seed ELISpot cultures with many more PBMCs in order to detect the true frequency and prevalence of circulating Ag-specific MBCs. Nevertheless, the similar magnitude of the MBC response to malaria Ags and to DT, a protein of fairly similar length to the malaria Ags used, implies that mounting a B cell response to malaria Ags is not necessarily any more difficult than mounting a B cell response to other Ags. Although it is possible that numbers of DT-specific MBC may have been much higher immediately after vaccination in infancy, the lack of any detectable decline in DT-specific MBC frequency with increasing age tends to argue against this possibility.in vivo upon encounter with an Ag. Moreover, for different types of Ags the efficiency of the differentiation of MBC into ASC may be different. A direct comparison of MBC frequencies derived from the ELISpot assay with MBC precursor frequencies derived from limiting dilution assays or from antigen-specific flow cytometry (using chromophore-labled antigen) is urgently needed to validate the accuracy and sensitivity of the B cell ELISpot assay.Another caveat of the B cell ELISpot assay is that we do not know whether the stimulation medium differentiates MBC into ASC at a fixed ratio or whether this is a true reflection of what happens Related to this is the question of whether B cell ELISpot data should be presented as the proportion of all MBCs that are specific for the Ag of interest (% MBC) As in most studies of human immune responses, access to the tissues of real interest - in this case spleen and bone marrow - is restricted and observations from studies of PBMC need to be interpreted with caution. In our study, the presence of detectable Abs and MBC did not correlate for any of the Ags tested, and a significant proportion of individuals lacked detectable circulating MBCs despite having significant titres of the corresponding plasma Ab. While this may reflect insufficient sensitivity of the assay, it is also highly plausible that ASCs present in tissues (either as short-lived plasma cells (SLPC) in spleen or as SLPC or long-lived plasma cells (LLPC) in bone marrow) continue to secrete Abs even though the frequency of circulating MBCs has fallen below the lower limit of detection of the ELISpot assay. A similar lack of correlation between MBC and serum Abs was observed in the Thai study P. falciparumB cell mitogenic activity has long been ascribed to The functional significance of the relatively high frequencies of cells with the phenotype of so-called “atypical MBCs” in malaria-exposed individuals is not known. One potential explanation is that these cells - which have a substantially shorter life span than classical MBC In summary, although the prevalence of individuals with malaria-specific MBCs increases with increasing age  the magnitude of an individual's malaria specific MBC response is similar in children with minimal prior malaria exposure to adults with a considerably higher cumulative malaria exposure. However, the antigenic repertoire of the B cell response is more limited in children than in adults and only increases with increasing exposure. Since the magnitude of the malaria-specific B cell response is similar to that induced by diphtheria vaccination, we find no evidence that mounting a B cell response to malarial antigens is more difficult than to other antigens. Nevertheless, the possibility remains that the development and/or retention of malaria specific memory B cells is more efficient in areas of low malaria transmission, such as The Gambia or Thailand, than in very highly endemic areas such as Mali. If so, this might be explained either by competition for limited numbers of environmental niches for MBC survival, or by preferential induction of SLPC rather than LLPCs, in frequently infected individuals.The studies were approved by both the Gambia Government/Medical Research Council (MRC) Joint Ethics Committee and the Ethics Committee at the London School of Hygiene and Tropical Medicine. Participants were enrolled after individual written informed consent was obtained from the participant or their parent/guardian.Blood samples from 118 healthy individuals were collected in May–June 2009 (prior to the transmission season) from Brefet, Foni District, The Gambia. Brefet is a rural village situated 55 km inland from the Atlantic coast and 1 km from the river Gambia. In The Gambia, malaria transmission is seasonal, starting in July and ending in December with the peak of transmission being in November Villagers were grouped according to age into the following six age categories  and 20 individuals from each group were randomly selected. To avoid household clustering, all volunteers in each group received consecutive numbers, the total number of villagers per group was noted and out of this figure a random number generator drew 20 numbers to identify 20 individuals per age group. For individuals absent on the day of sampling, a new number was drawn. There was an equal distribution of males and females in each group.A venous blood sample of 5 to 20 ml was obtained from each participant in accordance with the age-specific guidance provided by the Gambian Government / MRC Joint Ethics Committee. A thick blood film was prepared to test for presence of parasitaemia by slide microscopy, 0.5 ml of blood was collected into EDTA tubes for parasite detection by PCR and the remainder of the sample was collected into heparinised tubes and used for B cell ELISpot and Ab measurement by ELISA. HIV testing was not done on any of the samples, and HIV status was not reported. Adult HIV prevalence in rural areas of The Gambia is estimated to be approx 2% in 2009 A village health worker was present in Brefet throughout the transmission season to diagnose malaria infections by use of RDT (OptiMAL®) for malaria. Study participants had been asked to report to the village health centre if they suspected they had malaria. Anti-malarial treatment was only given if the rapid diagnostic test was positive.At the end of the transmission season (December 2009), a finger prick sample from each study participant was obtained to assess i) anti-malarial Ab levels, and ii) parasitaemia levels by PCR. Samples from 20 individuals were missing (18 had travelled away from the village and 2 had withdrawn their consent). A week later, a further venous sample for B cell ELISpot was requested from those individuals who showed a≥1.5 fold increase in Ab concentration for at least 1 Ag in comparison to the value recorded at the start of the malaria season in May and/or for whom a clinical episode of malaria during the last transmission season was confirmed by a positive RDT. Individuals with increased Ab concentrations and/or a clinical episode of malaria are collectively referred to as cases. Age-matched (plus/minus 1 year) controls  were selected from the study cohort for comparison.For the measurement of AMB, blood collected from children enrolled in an ongoing malaria case-control study based in the coastal area of The Gambia 19 and MSP-2 Ags used were glutathione s-transferase (GST) fusion proteins representing amino acids 1631–1726 of the Wellcome allele of MSP-11919 is highly conserved among P. falciparum isolates with minor sequence variations having a minimal effect on antibody recognition The MSP-16 PBMCs in RPMI containing 10% fetal calf serum, 100 U/ml penicillin, 100 ug/ml streptomycin and 2 mM L-glutamine  were added to each well of a 24-well culture plate, and supplemented with 50 µM β-Mercaptoethanol, 0.5 µg/ml Phytolacca Americana pokeweed mitogen , Staphylococcus aureus Cowan , 3 µg/ml CpG-2006 (Eurofins MWG-Operon- 5′TCG TCG TTT TGT CGT TTT GTC GTT 3′) and 25 ng/ml recombinant human IL-10 (R&D Systems) and placed in the incubator  for six days. On day 5, quadruplicate wells of multiscreen-HA plates  were coated overnight with either unbiotinylated AffiniPure F(ab′)2 fragment donkey anti-human IgG (Jackson ImmunoResearch Laboratories), or with one of the four malarial Ags, DT or KLH, respectively. On day six, 400,000 cultured cells were added to each antigen coated well (2000 cells for IgG coated wells), and incubated for 6 hours . Cells were then incubated overnight (at 4°C) with biotin-SP-conjugated AffiniPure fragment donkey anti-human IgG (Jackson ImmunoResearch). The next day, strepavidin-AKP (BD Biosciences) followed by detection solution (AP conjugate substrate kit (BioRad)) was added and plates were read using the AID ELISpot reader. All samples in the IgG coated wells yielded more than 1000 spots/million PBMC, a threshold previously defined for a valid assay PBMC were isolated from the participant's whole blood as described elsewhere Only values above the upper limit of the 99% confidence interval of the median obtained for the negative control KLH (0.004% or more than 1.88 spots/million PBMC) were considered as positive responses.19, MSP-2, MSP-3 and AMA-1 were measured by ELISA, using the method described elsewhere Concentrations of IgG binding to MSP-1A positive response was defined by a value above the upper limit of the 99% confidence interval obtained for this Ag with a pool of plasma samples obtained from 20 malaria naïve tourists from Europe. Only individuals which were above the cut-off value were termed responders.Ab levels to DT were quantified using a commercial kit  following the manufacturer's instructions. OD values are converted to International Units (IU/ml) by comparison with the standard curve. The manufacturer considers antibody titres >0.1 IU/ml sufficient for protection.Plasmodium falciparum ribosomal DNA was performed as described previously DNA was extracted from 150 µl of EDTA blood using the X-tractor GeneTM robot, according to the manufacturer's instructions (Corbett Robotics) +CD21−CD27−CD10−. All samples were acquired on a 9-colour CyAn ADP flow cytometer and were analysed by FlowJo software .Surface staining of AMB was performed on freshly isolated PBMC. The cells were stained with the following fluorochrome-labelled mouse anti-human antibodies: ECD-anti-CD19 (Beckman Coulter), and APC-anti-CD21, APC-Cy7-anti-CD27, Pe-Cy7-anti-CD10 . AMB were identified as follows: CD19Stool samples were collected into fixative  and processed using the ParasiTrap fecal diagnostic system (Biosepar) and read by the routine microbiology laboratories at the MRC in Fajara, The Gambia.Data were analysed using GraphPad Prism 5 and STATA 10.1. Chi-squared tests for trend were used to assess whether the percentage of individuals having Ab and MBC changed significantly with age for each Ag as well as for measuring the breadth of the immune response. Kruskal-Wallis tests were used to assess whether Ab concentration or MBC numbers differed significantly between age groups, and to test for differences in MBC numbers for the different Ags. As appropriate, Wilcoxon matched paired tests or Mann-Whitney tests were performed to compare two groups. Where multiple tests were performed on the same individuals for multiple responses to different malarial Ags, p values were adjusted for multiple comparisons using the Bonferoni correction. To assess the agreement between the two read outs used for the ELISpot results (% MBC or MBC/PBMC) in identifying “responders” Kappa statistics were calculated, and the results graded using the classification by Landis & Koch Table S1Baseline characteristics of the study cohort.(DOC)Click here for additional data file.


Malaria remains the leading communicable disease in Ethiopia, with around one million clinical cases of malaria reported annually. The country currently has plans for elimination for specific geographic areas of the country. Human movement may lead to the maintenance of reservoirs of infection, complicating attempts to eliminate malaria.An unmatched case–control study was conducted with 560 adult patients at a Health Centre in central Ethiopia. Patients who received a malaria test were interviewed regarding their recent travel histories. Bivariate and multivariate analyses were conducted to determine if reported travel outside of the home village within the last month was related to malaria infection status.Plasmodium falciparum  but not for infection with Plasmodium vivax . Male sex was strongly associated with any malaria infection .After adjusting for several known confounding factors, travel away from the home village in the last 30 days was a statistically significant risk factor for infection with Given the importance of identifying reservoir infections, consideration of human movement patterns should factor into decisions regarding elimination and disease prevention, especially when targeted areas are limited to regions within a country. Malaria is the leading cause of morbidity in Ethiopia, with more than one million clinical cases of malaria reported annually . ControlPlasmodium falciparum malaria infection in refugee settings, coastal areas, urban areas and river basins [There is evidence to suggest that human movement, interacting with vector habitat and features of the environment may be important for the epidemiology of malaria ,12. Peopr basins -16, reinr basins . This imr basins ,18.As malaria transmission continues to fall in many parts of Africa due to the continued scale-up of malaria prevention and control efforts, transmission is likely to become increasingly heterogeneous, disproportionately affecting sub-populations in high-risk communities . This coThis study explores the role of travel history as a risk factor for malaria parasite infection. Investigating aspects of human movement is applicable to Ethiopia and other settings targeting elimination, as it focuses on identifying factors associated with being a source case of infection.Plasmodium infection, regardless of parasite species. Controls were persons presenting at the same facility whose thick blood smear or RDT tests results were negative for infection.A health facility-based, unmatched case–control study was conducted from 22 June, 2011 to 14 October, 2011. Cases were persons presenting at the Bulbula health facility   where altitude ranges from 1,500-2,300 m above sea-level. The health centre is located in a market town with a current population of ~8,500 and provides primary health care services to outpatients and serves as the central point of the primary health care system in the area [teff, maize, and vegetables. Agricultural opportunities have also attracted significant numbers of migrant workers to the area [P. falciparum and Plasmodium vivax infections . Anopheles arabiensis is the main malaria vector in the country [Bulbula health centre is a malaria epidemic detection and sentinel surveillance site in Oromia, part of an on-going collaboration between the U.S. Presidents Malaria Initiative, the Ethiopia Health and Nutrition Research Institute, and the Oromia Regional Health Bureau. The health centre is located in Adami Tulu Jido Kombolcha the area . The heathe area . Surveil country ,22.wereda, fever or history of fever in the previous 72 hours, and received a Giemsa stained thick blood smear or RDT for malaria diagnosis at the health centre on the day of recruitment.Inclusion criteria for the study were: attendance at the Bulbula Health Centre Out Patient Department (OPD), age 18 years or older, residence in Adami Tullu Jido Kombolcha wereda.Participants were excluded from the study if they were under 18 years of age, extremely ill and in need of immediate medical attention at the time of testing, had self-reported malaria in the previous 30 days (excluding the current episode), had taken malaria prophylaxis or treatment in the previous 30 days, or were not full-time residents of Adami Tullu Jido Kombolcha Sample size calculations were conducted in the StatCalc module of EpiInfo . The study was powered to detect an odds ratio of 2.0 with 95% significance and 80% power on the measure of having travelled and spent a night away from the home (outside the immediate community) in the previous 30 days, assuming that 20% of those who were not infected with any parasite species had travelled overnight in the past month, and a 1:4 case–control mix. In total, 560 patients were recruited for the study. These consisted of 141 cases  and 419 controls .Participants were recruited over a 16-week period. After obtaining informed consent, patients were interviewed using a standard, pre-tested questionnaire covering socio-demographic and household risk factors for malaria including recent travel history. Interviews were conducted in either Oromiffa or Amharic, depending on the language preference of the patient. To minimize recall bias, an Ethiopian calendar was supplied for use as a reference for recalling travel dates. When recording the names of the villages where patients travelled, the data collector used a list of kebeles with corresponding village names provided by the Ethiopian Central Statistics Agency and a map of the surrounding area. Patients were first asked about any overnight stays away from their home village in the past 30 days, then respectively asked to identify when and where they had travelled and from which dates with the aforementioned aides. After the interview was complete, the patients were taken back to the laboratory to obtain their malaria test results; participants were then classified as either a case or a control. Data were double entered in Microsoft Access  database and converted to STATA 10.0  format for analysis.2), Fischer’s exact tests, and multivariate logistic regression analysis was conducted to test whether travel was a risk factor for malaria. Multivariate logistic regression analysis was used to control for age, sex, socio-economic status and use of protection methods such as bed nets. Sub-analyses of cases identified as P. falciparum infection vs no P. falciparum infection, and P. vivax infection vs no P. vivax infection were conducted. Mixed infections (n=1) were included as cases in both sub-analyses. A simple wealth index was created using binary variables for durable asset possession using principal components analysis (PCA) [Chi-square (χis (PCA) . Only this (PCA) ; the patInstitutional Review Board (IRB) approval was obtained from Tulane University in New Orleans, Louisiana, USA and the IRB of the Addis Continental Institute of Public Health in Addis Ababa, Ethiopia.P. falciparum or P. vivax malaria, respectively; only one patient had a mixed infection.A total of 560 patients were successfully recruited for the study: 141 cases and 419 controls. Of the 141 infected individuals, 85 (60.3%) and 57 (40.4%) were diagnosed with vs 44.7%)  Table . The agePatients self-reported that their homes were primarily roofed with thatch/leaf or corrugated iron, while wall structures were largely reported to be covered with adobe or wood. Adami Tulu Jido Kombolcha is a largely rural area, where most people are engaged in farming and other agricultural activities as a means of subsistence, as such, 87% of study participants report owning animals, largely cattle, donkeys or sheep.vs 30%; Odds Ratio (OR)=1.65, p=0.005). Most trips (67%) were made to areas within Adami Tulu wereda. Travel history was associated with malaria infection, but the relationship was only of borderline significance, regardless of infecting parasite species.One hundred ninety four study subjects (35%) reported staying overnight outside of their home village for at least one night during the previous 30 days. A total of 203 overnight trips were reported by these persons. Nearly all travellers made only one trip overnight during the previous 30 days. Among those reporting an overnight stay, the median length of stay was three days (Interquartile range 1–28 (mean 5.2 days)). Travel was more common among men than among women .Two hundred and thirty four (41.8%) study participants reported that their household had at least one ITN. Of those who reported owning an ITN, 108 (46.2%) reported having slept under an ITN the night before the interview, meaning that overall, only 19.3% of all study participants had used a net the night before they enrolled in the study. Only 5% of patients with a history of travel reported ITN use during their travel. Sleeping under an ITN did not appear protective against malaria in this population and was not associated significantly with being either a case or control . Patients who lived in houses with corrugated iron roofs were less likely to be infected with P. falciparum than those living in houses with other roof types . No statistically significant relationship of roof type with P. vivax infection was seen . Having a wall type other than covered adobe appeared to be a strong predictor of P. vivax , but not for P. falciparum infection.The vast majority (519 or 93%) of laboratory confirmed malaria infections were confirmed with microscopy, the remainder of laboratory confirmations (41 or 7%) used RDTs. No significant differences were found between the odds of infection using either method. Men were more likely to be diagnosed with malaria of all types than women , and P. falciparum, P. vivax and all malaria cases as outcome variables to adjust for potential confounding. Known and measured potential confounders included sex, age, socio-economic status, household characteristics, and use of preventative measures (ITNs); all variables were included in the final logistic regression models. The results of the analysis are shown in Table P. falciparum diagnosis were more likely to have travelled in the previous 30 days than controls (Adjusted Odds Ratio (AOR)=1.76, 95% CI: 1.06–2.93). This was also true for all malaria cases , but not for P. vivax infections alone . Age appeared to be protective for P. vivax, but not for P. falciparum infection, while wealth was associated with reduced odds of P. falciparum infections alone. Male sex, even after adjusting for travel, household factors, SES and use of a protective measure, was associated with P. vivax infection .Logistic regression models were fit with P. falciparum infection even when the travel occurs within endemic areas. Such endemic areas, including the area where this study was conducted, may vary in risk of infection even within relatively small geographic zones, for reasons which may related to environmental factors as well as a myriad of other influences [While travel to a malaria-endemic area in Ethiopia is recognized as a risk factor for malaria infection, much of the country is endemic for malaria transmission and little attention has been paid to whether or not routine human movement patterns can lead to higher risks of malaria infection, even on a small geographic scale within endemic areas. This study shows that travel may be a risk factor for symptomatic fluences ,18. ThusIn this study blood slides and RDTs were performed only once, as such misclassification bias could arise due to the potential for inaccurate microscopy readings by facility technicians. However, the facility was part of an on-going quality assurance and control programme (QA/QC) at the time of the study. The contemporaneous QA/QC results showed 96% agreement of facility readings with expert readings over the five month period surrounding the study with regards to positivity and negativity, and 90% agreement on species identification. This suggests that any misclassification bias associated with microscopy conducted at the facility level is likely to be small, furthermore, such a bias would likely be in the direction of the null hypothesis.P. falciparum infections, even within moderately endemic areas of the country  [Within low transmission areas, importation of infections could lead to maintenance of reservoirs of infection in the absence of local vectorial capacity to maintain transmission ,26. Undetely 7%) .P. vivax possesses a dormant hypnozoite stage and causes relapses of malaria, many of the P. vivax infections in this study may have originated not from initial sporozoite inoculations, but rather as relapses. The epidemiology of the disease appears different among P. vivax infections in this study, travel, at least recent travel, was not a marked risk factor, while age and sex were important. In this study male sex was the greatest risk factor for all categories of malaria infection. It is possible that behavioural factors associated with being male  could be predisposing towards malaria infection. While men in this study were more likely to travel than women, and spend the night away from their home village, even after adjusting for this travel, sex remained a significant risk factor for all types of malaria infection.As P. falciparum, and was associated with increased infection with P. vivax. While this finding is out of line with the literature and large-scale evaluation research, lack of protection from net use has been reported in observational studies in Ethiopia before [P. vivax infection, endogeneity between the use of nets and malaria risk or simply the limited sample size.In this study, ownership or use of an ITN showed no statistically significant protective effect against a before -31. In tIn order to assess effect-modification through the relaxed use of protective measures during travel the interaction between net use and travel was tested. Though the interaction term was statistically insignificant in this context, this was possibly hampered by small sample size. Regardless, the low level of use of LLIN use indicates the need to focus on developing a strong distribution system to maintain LLIN coverage over time, and to increase use among those travelling and staying away from their home overnight.P. falciparum infection, the study is a small case–control study of limited geographic scope. Because this study focused only on patients presenting at one health centre, the study has limited external validity. Data collection from national parasitemia surveys or at a minimum from multiple health centres would help to make these results more generalizable. Further, the size of the adjusted odds ratios found indicate that travel is not a strong risk factor. However, given that the study area has higher transmission than some other areas of Ethiopia, notably the Rift Valley basin near Eritrea or highland areas, travel may be an even more important factor in other areas of the country [While this study indicates travel is a risk factor for  country . Travel P. vivax is generally poorly explored [P. vivax transmission is likely to be more resilient to interventions that reduce transmission pressure for several reasons including greater and more rapid production of gametocytes, shorter extrinsic incubation periods and the dormant liver stage [P. vivax infections in this setting but could easily contribute to the maintenance of transmission even in very low transmission areas.The epidemiology of explored  and giveer stage . As suchDerivation of travel history from individual recall is likely to be subject to recall bias. Recent technological developments including inexpensive small Global Positioning System data loggers and mobile phones may give rise to the ability to correct for these biases to some extent, by allowing for relatively fine scale temporal and spatial tracking of human or at least device movement ,34. The These findings suggest that careful consideration should be given to travellers and perhaps especially adult men in areas of moderate transmission in Ethiopia. These individuals appear to be at the highest risk of malaria infection. While this study cannot determine if the risk evolves from travel to areas of higher risk or relaxation of behavioural preventative measures during travel, it indicates the potential for interventions on communicating the importance of maintaining behavioural prevention activities, such as use of bed nets during travel. It additionally suggests that such communication measures might be most advantageous if targeted towards men.P. falciparum infection in a highland fringe area of Oromia, Ethiopia while male sex is a risk factor for all types of malaria infection. Men travel more frequently overnight than women, importation of infections may be an important contributor to maintenance of transmission even in relatively high transmission areas for Ethiopia. Further investigation quantifying the role of importation of infections to endemic areas could contribute to programmatic strategies to locally interrupt transmission in Ethiopia.Travel is a risk factor for The authors declare that they have no competing interests.JY, JK and CT conceived the study, developed the methods, conducted the analysis and drafted the manuscript. TE and RR contributed to study development and drafting of the manuscript. HN, YB and CT contributed to the development of study methods, collected the data, and critically read and contributed to the drafting of the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum sexual stages, induces also a dose-dependent haemolysis, especially in glucose-6-phosphate dehydrogenase deficient (G6PDd) individuals. Therefore, it is important to determine the prevalence of this deficiency in areas that would potentially benefit from its use. The prevalence of G6PD deficiency by genotype and enzyme activity was determined in healthy school children in The Gambia.Primaquine, the only available drug effective against Blood samples from primary school children collected during a dry season malaria survey were screened for G6PDd and malaria infection. Genotypes for allele mutations reported in the country; 376, 202A-, 968A- and 542 were analysed while enzyme activity (phenotype) was assayed using a semi-quantitative commercial test kit. Enzyme activity values were fitted in a finite mixture model to determine the distribution and calculate a cut-off for deficiency. The association between genotype and phenotype for boys and girls as well as the association between mutant genotype and deficient phenotype was analysed.P. falciparum malaria infection was 14%. The prevalence of the 202A-, 968 and 542 mutations was 1.8%, 2.1% and 1.0%, respectively, and higher in boys than in girls. The prevalence of G6PDd phenotype was 6.4% , 7.8% (57/728) in boys and 4.9% (35/709) in girls with significantly higher odds in the former . The deficient phenotype was associated with reduced odds of malaria infection .Samples from 1,437 children; 51% boys were analysed. The prevalence of There is a weak association between genotype and phenotype estimates of G6PDd prevalence. The phenotype expression of deficiency represents combinations of mutant alleles rather than specific mutations. Genotype studies in individuals with a deficient phenotype would help identify alleles responsible for haemolysis. Plasmodium falciparum with the aim of interrupting transmission, is of particular interest[The reduction in the malaria burden observed in several endemic countries has revived optimism about the possible elimination of this disease-3. In thinterest. Currentinterest. Neverthinterest,7. ThereG6PDd is considered the most common enzyme deficiency, affecting about 5% of the world’s population, though in most cases it is clinically silent. It is cG6PD status is determined by molecular and/or biochemical methods. ScreeniThis study presents an update on G6PD genotype frequencies reported in The Gambia and describes the association between genotypes and phenotypes (enzyme activity) in healthy school children.As part of a malariometric survey, blood s340nm/∆time)/OD405nm.Enzyme activity was measured using a commercial quantitative ELISA kit . It is based on the spectrophotometric measurement of reduced nicotinamide adenine dinucleotide phosphate (NADPH) released during G6PD enzyme activity and, colorimetrically by reduction of tetrazolium salt (550 nm). The readings from the 340 nm measurement were used in the calculations. A 6 mm diameter disc was punched from each dried blood spot (DBS) into a U bottom microtitre plate and eluted with 75 μL of elution agent. NADPH was measured as absorbance at 340 nm in kinetDNA was extracted using protocols for the QiaExtractogen liquid handling robot (QiaGen). Allele polymorphisms were detected by real-time PCR with TaqMan® SNP genotyping assays using ABI PRISM SDS 7500 . Oligonucleotide primers and probe genotyping assay mixes for nucleotides A376G, G202A, A542T and T968C, were also obtained from Applied Biosystems. Genotyping assay were performed in a final reaction of 25 μL, containing 12.5 μL of 2× TaqMan Universal Master Mix, 0.5 μL of 40× TaqMan SNP genotyping assay mix and at least 10ηg of genomic DNA in 11 μL of distilled water. The amplification conditions were 2 minutes at 50°C for AmpErase uracil-N-glycosylase activity and 10 minutes at 95°C for Amplitaq Gold activation, followed by 40 cycles of 15 seconds at 95°C for denaturation and 1 min at 60°C for annealing and extension. The samples were run together with the non-template control. Allelic discrimination was performed on fluorescence data using the ABI Prism 7500 allelic discrimination software and the free TaqMan-Genotyper Software . Each sample result was verified by examining the PCR curve generated to eliminate false-positive results due to aberrant light emission.2. A logistic regression model was used to determine the relationship between allele mutation and a deficient phenotype and the association between haemoglobin, gender and parasitaemia on deficient phenotype. Data was analysed using Stata 12 (College Station Tx).Children were classified on the basis of allelic discrimination patterns as normal, mutant (hemi- or homozygous) or heterozygous  while genotypes were defined for each of the polymorphic A variants; 376, 202, 968, and for the 542 variant. The distribution of G6PD enzyme activity in the sample was analysed using a two-component finite mixture model. Using the population with the higher mean activity as reference, the cut-off for activity in the sample was defined as < −2SD below the mean of this subpopulation. The association between G6PD activity and allele mutation in males and females was examined using Pearson χP. falciparum) determined by PCR was 14%[Blood samples from 1,437 children; 728 (50.7%) of them boys were analysed. The mean age was 10 years (SD 2.3) and mean haemoglobin was 12.4 g/dl (SD 1.3), with no difference between males and females. The prevalence of malaria infection  and did not vary by gender however, the distribution in females showed two peaks of activity Figures a and b. The use of primaquine in malaria endemic areas should be balanced by the potential risk of haemolysis in G6PDd individuals hence the need for accurate estimates of the prevalence of deficiency.The 202A- variant is considered the most frequent in sSA so that the prevalence of G6PDd is often measured by detecting this mutation. Nevertheless, in this area the 968A- variant was more frequent than the 202A- variant, confirming previous reports,12 and iG6PDd has been associated with a protective effect against severe malaria in malesMeasuring single genotypes-30 does Two peaks were seen in the distribution of activity in females but not in males, which has been described with the more sensitive cytochemical assays,32. The For mass screening campaigns, qualitative testing is a feasible option as genotype methods are unlikely to be used routinely. However, newer methods, potentially adaptable for high throughput screening and addressing the limitation in females should be considered.The prevalence of G6PD deficiency and the risk of primaquine-induced haemolysis are important for deciding on the use of primaquine. The prevalence of G6PDd determined by measuring the phenotype (enzyme activity) is higher than that by genotype (mutations) analysis, though a substantial proportion of children are positive for both methods. Phenotype-based screening should be encouraged while genotype testing could be used to investigate subjects developing haemolysis but not identified as G6PDd by the qualitative tests.ACT: Artemisinin-based combination therapy; DBS: Dried blood spot; DNA: Deoxyribonucleic acid; G6PD: Glucose-6-phosphate dehydrogenase; G6PDd: Glucose-6-phosphate dehydrogenase deficiency; MRC: Medical Research Council Unit; NADPH: Nicotinamide adenine dinucleotide phosphate; PCR: Polymerase chain reaction; sSA: Sub-Saharan Africa.All authors declare that they have no competing interest.UDA and JO conceived and designed the project; JO lead the field work, analysed the data and wrote the draft manuscript. ENT designed the main study and supervised field work. SN, JP and MD processed the samples supervised by AAN, MA and DN. SJC contributed to the study design and supervised the field work; AAN analysed the G6PD sample data and reviewed the manuscript. UDA revised the manuscript text. All authors reviewed and approved the final manuscript.


As part of comprehensive malaria control strategies, the Bioko Island Malaria Control Project (BIMCP) distributed 110,000 long-lasting insecticide-treated nets (LLIN) in late 2007 with the aim of providing one net for each sleeping area. Despite attaining initially very high levels of net coverage and net use, many children under five years of age did not sleep under a net by 2009, according to annual malaria indicator surveys. The aim of this study was to assess the determinants of bed net use in children under five and bed net ownership of the households in which they live.Using data from annual cross-sectional household surveys of 2008 and 2009, we investigated factors associated with sleeping under a mosquito net the night prior to the survey, and a households owning at least one net, in all households which had at least one child under five years. Amongst others, caregiver's knowledge of malaria and household characteristics including a socio-economic score (SES), based on ownership of household assets, were analysed for their effect on net ownership and use.There was a decline of around 32% in the proportion of households that owned at least one net between 2008 and 2009. Higher household bed net ownership was associated with knowing how malaria was prevented and transmitted, having the house sprayed in the previous 12 months, having fewer children under five in the household, and children being sick at some point in the previous 14 days. Higher bed net use in children < 5 was associated with being sick at some point in the last 14 days prior to the survey, living in an urban area, more years of education of the head of the household, household ownership of at least one ITN (as opposed to an untreated net) and the year in which the survey took place.The big fall in bed net use from 2008 to 2009 was attributable to the striking decline in ownership. Although ownership was similar in rural and urban areas, rural households were less likely to protect their children with bed nets. Knowledge about malaria was an important determinant of bed net ownership. Further research is needed to elucidate the decline in bed net ownership between 2008 and 2009. Children under five years of age, together with pregnant women, have been identified as the most vulnerable risk group for malaria, with 88% of all deaths in sub-Saharan Africa attributed to malaria occurring in children under-five . In EquaInsecticide-treated nets (ITN) have been widely shown to be effective in reducing childhood morbidity and mortality through reducing mosquito bites while sleeping ,5. ITNs As has been previously reported, even when there are bed nets available in the household, children under five, the most vulnerable group, do not always have access to them -10. AlthIn a study conducted in five African countries , bed netVariables such as occupation and education levels of the household appeared to be directly associated with bed net use in some studies ,16. In EThe Bioko Island Malaria Control Project (BIMCP), which is funded by a consortium led by Marathon Oil Corporation and the Government of Equatorial Guinea, has conducted intensive malaria control activities in Bioko since 2004. These included free semi-annual rounds of IRS since 2004 targeting all households on the island, introduction of free artemisinin-based combination therapy (ACT) for pregnant women and children under 15 in 2005 (subsequently extended free-of-charge to the entire population in 2010), free Intermittent Preventative Treatment of pregnant women (IPTp) in 2005 as well as extensive information, education and communication (IEC) campaigns. The latter used mass media  as well as community-based interpersonal communications techniques. In 2007, the BIMCP distributed around 110,000 long-lasting insecticidal nets (LLIN) on Bioko Island free of charge. This intervention consisted of a door-to-door campaign with the intention to cover all sleeping areas in all households on the island. Householders were assisted with hanging nets where necessary. The mass distribution campaign resulted in very high levels of net ownership and use, but theses declined rapidly over time . All intThe objective of this study was to identify factors associated with use of bed nets in children under five in Bioko. As a secondary objective, this study aimed to identify the particular characteristics of the households that own bed nets.Data from two post-ITN mass distribution cross sectional annual surveys conducted by the BIMCP during February to April in 2008 and from August to September in 2009 respectively were analysed. The survey questionnaires were adapted from the Roll Back Malaria Monitoring and Evaluation Reference Group Malaria Indicator Survey and conducted in 18 sentinel sites across the island, as described elsewhere in more detail   according to a household census conducted by BIMCP . The surveys are, therefore, broadly representative of the entire population of Bioko of around 260,000 inhabitants .The original sample size of the surveys was calculated so that a change in prevalence of infection of 20% could be detected compared to the previous year at the same site with 80% power and a 5% type of 1 error level . In subshaving slept under any net the night before the survey" for each child under five, and "owning at least one bed net" for each household with a child under five. Most explanatory variables were collected at the household level, from the responses given by the person interviewed in the household, usually the head of household or caregiver. Data on knowledge of the person interviewed in the household was obtained through a series of questions concerning malaria prevention, malaria symptoms and malaria treatment . For the purpose of analysis, these questions were converted into three binary variables: "Is the use of bed nets an effective way to prevent malaria?", "Can mosquitoes transmit malaria?" and "Is fever a symptom of malaria?", each with a yes or no value.The outcome variables were "Other explanatory variables for the analysis of bed net use were: age and gender of the child, whether the house was sprayed with IRS in the last 12 months, disease status of each child (under-five) in the last 14 days (according to household interviewee), socio-economic status, age and education of head of household, type of eaves (open or closed), setting , existence of at least one ITN in the household. In this analysis only data on children under five living in households with at least one bed net (since a necessary condition to use a bed net is to own it). This dataset contained information relating to the individual child and data relating the household they lived in.Apart from the knowledge questions, the analysis on bed net ownership included the following explanatory variables: age and gender of the child, whether the house was sprayed with IRS in the last 12 months, disease status of each child (under-five) in the last 14 days (according to household interviewee), socio economic status, age, sex and education of head of household, setting  knowledge of availability of ACT for free, household size, living in the house for the last 12 months or more, and whether any woman in the household had been pregnant in the last year. In this analysis, data relating to households with children under-five were used (since bed nets are owned by households).et al , has meaOccupants of houses that were sprayed in the previous 12 months were more likely to own at least one bed net. These households could have been more sensitive to dangers of malaria, suggesting therefore both owning a net and having a house recently sprayed would be signs of enhanced awareness. Another explanation could also be that households sprayed in the previous 12 months were less remote and perhaps households with access to IRS are more likely to have access to other interventions, such as ITN delivery.Bed net ownership was directly associated with answering correctly two of the three questions assessing knowledge in the multivariable analysis. It seems reasonable that if caregivers knew how malaria was transmitted and prevented, they were more likely to protect their children with nets. This supports the notion as reported elsewhere ,35 that A surprising finding was that the greater the number of children under 5 in the household, the less likely it was that the household owned a bed net. It could have been that new households formed after the mass distribution in 2007 were more likely to have young children. This result needs further investigation since the under-five age group is of particular concern, even if the policy is one of universal coverage. Another unexpected finding was the association of being sick in the last 14 days and higher ownership. It could be that those children are perhaps sick more often and households retain their bed nets for longer periods, or it could be that they try to buy bed nets from the scarce sell points in the island, but this argument is thought to be unlikelyThe findings of this study must be viewed with some caution due to a number of limitations in the analytic methodology employed. First, as with all studies based on cross-sectional survey data assessments of causality are problematic and, therefore, all the conclusions presented here are what it is believed are the most plausible hypothesis to explain the results. Reverse causality is likely in some of the findings. For example in the association between ownership of nets and children being sick in the last 14 days, the sickness episodes could be a result of not being protected by a net because the household did not own one.Second, Principal Component Analysis was used to create a score as a proxy for socio-economic status. The extent to which this score represents the true socio economic status of a household was not validated for this particular setting. The variables included were a subjective choice by the authors based on previous literature of SES indexes, and therefore the final scores depend on the variables selected. As some authors already described , infrastFinally, it is possible that there was recall bias, especially with the questions regarding household sprayed in the last year, being sick in the last 14 days, education of head of household (in case the interviewee was not the head of household), and even whether the individual slept under a net the night before the survey.Bed net ownership was by far the factor most strongly associated with use in Bioko Island. The sharp decline in ownership from 2008 to 2009 was a striking finding of this study. If not reversed, this could undermine some of the progress previously made, as for example the two-thirds reduction in child mortality . There iThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Marathon Oil and other members of the funding consortium have no commercial interest in the findings made in this article.ALGB developed the study protocol, participated in the design and analysis, and drafted the manuscript. AR participated in the analysis of the paper and commented on the paper. IK supervised and conceived of the study and helped in drafting the manuscript. CS and GN coordinate the BIMCP and revised the manuscript. AM helped in the acquisition of surveys and drafted the manuscript. All authors read and approved the final manuscript.


Rapid diagnostic tests (RDT) are increasingly replacing microscopy for diagnosis of malaria in endemic settings. Although RDTs are simple and robust, errors in the post analytical phase i.e. in reading and interpretation of the RDT result, are not uncommon. In the Democratic Republic of the Congo (DRC) malaria is endemic, and malaria RDTs have been introduced since 2010. In June-July 2012, an external quality assessment (EQA) addressing correct reading and interpretation of the three band malaria RDT recommended by the National Malaria Control Programme was organized among end-users in DRC.High resolution photographs of 10 patient RDT results representing a variety of malaria diagnosis, (combinations of) species, invalid or unreadable tests, faint positive test lines etc. were prepared. Answers were encoded in multiple choice format. Photographs were sent to focal points in 9 out of 11 provinces in DRC, who distributed them to malaria RDTs end-users. For each health facility, 1 questionnaire on availability, training and use of RDTs in the structure accompanied the photographs. End-users were requested to answer the multiple choice individually, by sending a short text message (SMS) to the study coordinator in Kinshasa who transferred SMS by blue tooth to an excel database. Questionnaires were recollected from the health facility by the focal points and sent back to Kinshasa.Plasmodium falciparum infection. Failure to detect faint or weak test lines was a common reason for wrong answers.Preliminary results are presented. In total, more than 2000 end-users participated in this EQA. Overall, about one out of 5 participants red and interpreted all 10 photographs correctly. Less than 1% had all 10 answers incorrect. For each individual photograph, between 50-90% of correct answers were received. In up to 40% of answers, the result represented a major error such as not recognizing an invalid test result, not recognizing negative test results or not recognizing a The current EQA consisted of an innovative combination of a photograph-based approach, as being used in HIV and malaria RDT trainings, with communication by standard cell phone and SMS. Using this approach, we confirmed that errors in reading and interpretation of malaria RDTs are widespread in DRC. Data generated through this study can be exploited by control programs to identify common problems in job aids or end-user’s performance and to organize appropriate remediation. Organisation of regular external quality assessments with feed-back to the participants might as well improve end user performance by its educational stimulus. In addition, recording of basic professional data through EQA, allows future access to the end-users by cell-phone network for malaria RDT-related issues.


Background. Helminth and malaria coinfections are common in the tropics. We investigated the hypothesis that prenatal exposure to these parasites might influence susceptibility to malaria in childhood.Methods. In a birth cohort of 2345 mother–child pairs in Uganda, maternal helminth and malaria infection status was determined during pregnancy, and childhood malaria episodes were recorded from birth to age 5 years. We examined associations between maternal infections and malaria in the offspring.Results. Common maternal infections were hookworm (45%), Mansonella perstans (21%), Schistosoma mansoni (18%), and Plasmodium falciparum (11%). At age 5 years, 69% of the children were still under follow-up. The incidence of malaria was 34 episodes per 100 child-years, and the mean prevalence of asymptomatic malaria at annual visits was 5.4%. Maternal hookworm and M. perstans infections were associated with an increased rate of childhood clinical malaria . S. mansoni infection had no consistent association with childhood malaria.Conclusions. This is the first report of an association between helminth infections in pregnancy and malaria in the offspring and indicates that helminth infections in pregnancy may increase the burden of childhood malaria morbidity. Plasmodium and helminths causes an enormous public health burden in the tropics [Plasmodium falciparum may increase malaria risk in early childhood [Chronic infection with  tropics –5. Malar tropics , 7. Recehildhood , 9; howe–helminth coinfections on childhood malaria have not been addressed.In the 1990s, estimates suggested that 44 million of the world's pregnant women harbored hookworm , and it Plasmodia may possibly modulate responses to helminth coinfections [Studies including the EMaBS have reported associations between malaria and helminth infections in pregnant women , 16, andfections . Prenatafections  or the dfections , respectfections , 29.In the EMaBS, treatment with anthelminthics in pregnancy had no effect on childhood malaria incidence. However, we speculated that the single-dose treatment might be insufficient to change an effect of helminths established earlier during pregnancy. This study used EMaBS data (ISRCTN32849447)  to examiThe study was approved by the Science and Ethics Committee of the Uganda Virus Research Institute, the Uganda National Council for Science and Technology and the ethics committee of the London School of Hygiene and Tropical Medicine. Project staff explained the study to the participants in the local language and provided participants with a study information letter to take home. Written informed consent was obtained from the mother during pregnancy and from the mother or caregiver when the child was aged 1 year.P. falciparum is the dominant Plasmodium species; Anopheles gambiae and Anopheles fenestus are the dominant vectors [The study area is on the northern shores of Lake Victoria in Uganda, a high malaria transmission area.  vectors . The areThe EMaBS enrolled 2507 pregnant women in their second or third trimester between April 2003 and November 2005; 2345 live births were recorded. Inclusion and exclusion criteria are described elsewhere . We presP. falciparum and helminth infections and were treated according to clinical guidelines if infections were found.At enrolment, the median gestational age was 27 weeks . Before receiving the trial intervention, women gave a blood and stool sample for assessment of parasite infections. Women were then randomized to single-dose albendazole (400 mg) or placebo, and praziquantel (40 mg/kg) or placebo; their offspring were randomized to receive quarterly single-dose albendazole (200 mg from age 15 to 21 months and 400 mg from age 24 to 60 months) or placebo. Ferrous sulphate was provided monthly, and intermittent presumptive treatment for malaria (sulphadoxine-pyrimethamine) twice during the pregnancy. All women received anthelminthic treatment 6 weeks after delivery. After delivery, mothers were invited to bring the children to the research clinic for routine immunizations and any illness and for quarterly study visits to age 5 years. Community workers visited the children fortnightly and referred sick children to the clinic. Clinical malaria episodes were recorded prospectively. At annual scheduled visits, the children were examined for Participants' addresses at enrollment were geo-referenced using handheld GPS receivers, and geographical zones were established based on features such as coastline, forest, location of settlements, and altitude .Mansonella perstans using the modified Knott's method [Women provided blood and stool samples at enrollment and after delivery; children provided samples at illness and routine annual visits. Blood samples were examined for s method . Thick bs method , 34. Hums method ; for infP < .05, assuming that each intervention had an independent effect. Either direction of effect could happen, depending on whether helminth coinfection increases susceptibility to malaria infection and disease or decreases inflammation and hence reduces malaria-induced morbidity.The aim was to examine the association between maternal helminth and malaria infections in pregnancy and malaria in the offspring. The sample size for the study was determined for the original trial objectives. To test the hypothesis that maternal albendazole or praziquantel in pregnancy would influence the incidence of malaria in infancy , a study with 2500 participants would have 80% power to show an 18% reduction or a 19% increase in the incidence of malaria in infancy, with P. falciparum parasitemia as determined at annual visits to age 5 years. Childhood clinical malaria was defined as a history of recent fever or axillary temperature of ≥37.5°C and any parasitemia. Asymptomatic P. falciparum parasitemia was defined as a positive malaria slide in the absence of fever on the sampling day. Key predictor variables were maternal P. falciparum, hookworm, M. perstans, and S. mansoni infections at enrollment. Malaria was defined as peripheral parasitemia.Of 2345 live births, 53 twins and 3 triplets were excluded, leaving 2289 children for inclusion in the analysis. Primary outcome was incidence of childhood clinical malaria from birth to age 5 years, and secondary outcome was prevalence of asymptomatic P ≤ .10) at the univariable analyses were entered into multivariable models. Variables included in the models were maternal age, education, parity, HIV status, mosquito net ownership, socioeconomic status, and geographical residential zone. Maternal P. falciparum and HIV infections and child albendazole were assessed as potential effect modifiers of the association between each maternal infection and childhood malaria. The secondary outcome was analyzed by combining data from all annual visits and comparing repeated prevalence of parasitemia between maternal malaria and helminth infection groups using random effects logistic regression, adjusting for the same confounders. Adjusted P values were calculated using likelihood ratio tests. Albendazole and praziquantel treatment in pregnancy had no effect on the incidence of clinical malaria [P values ≤ .05.For the primary outcome, time at risk began at birth and was censored at loss to follow-up, death, or age 5 years. Clinical malaria episodes within 14 days of an initial presentation were regarded as a recrudescence and excluded from the analysis; time at risk was adjusted accordingly, excluding these 14-day periods from the total person-time denominator. Crude hazard ratios (HRs) for the effect of malaria and helminth infections in pregnancy on the incidence of childhood malaria were calculated using Cox regression with robust standard errors to allow for within-child clustering of malaria episodes. Independent risk factors for maternal infections and childhood malaria that were significant . Table Supplementary Appendix 1. The overall mean P. falciparum parasite count in pregnancy was 163 , and only 51 (22%) of 236 mothers with parasitemia had >1000 parasite/µL blood. Two hundred two (8%) women were infected with P. falciparum and at least one of hookworm or S. mansoni or M. perstans. At delivery, only 69 of 2133 (3%) of the women tested had malaria parasitemia. Maternal HIV prevalence was 11%.Of 2345 live births, 1622 children (69%) were still under follow-up at 5 years, and a total of 33 178 clinic visits for illnesses were recorded . A trialThe prevalence of helminth infections was low among the children: 2.9% , 5.2% , 7.7% , 9.0% , and 9.5%  at annual visits 1, 2, 3, 4, and 5 years, respectively. Of 2289 children, 1161 (51%) had at least 1 malaria episode; 459 (20%) had 1 episode, and 702 (31%) had ≥2 episodes (18 children had >10 episodes). The overall malaria incidence rate was 34 per 100 child-years, higher in the first 2 years , than in years 3, 4, and 5 . The annual prevalence of asymptomatic parasitemia among the children was 5.9% , 7.1% , 4.7% 95% CI, (3.7–6.0), 4.3%  and 4.8%  at 1, 2, 3, 4, and 5 years, respectively. The overall mean parasite density was 8292 parasites/µL .M. perstans infections were associated with increased odds of maternal malaria. The risk of maternal helminth and malaria infection in pregnancy varied significantly by geographical zone [Older age, higher education, higher socioeconomic status, and mosquito net ownership were associated with a lower prevalence of maternal helminth and malaria infections . HIV infcal zone .Table P = .04). However, the positive association observed between maternal malaria and childhood asymptomatic parasitemia did not reach statistical significance . Infection intensity did not affect the association. The association was not modified by maternal HIV (Pinteraction = .20) or child albendazole (P = .60).After adjusting for maternal age, parity, education, mosquito net ownership, household socioeconomic status, maternal HIV status, and location of residence, maternal malaria was associated with a significantly higher incidence of childhood malaria  or childhood asymptomatic parasitemia  but weakened the association between maternal M. perstans and childhood clinical malaria  or childhood asymptomatic parasitemia . Overall, there was no association between maternal S. mansoni infection and childhood clinical malaria or asymptomatic parasitaemia (Tables P = .10), M. perstans (P = .80), or S. mansoni (P = .80). Similarly, child albendazole did not modify the associations between childhood malaria and maternal hookworm (P = .30), M. perstans (P = .10), or S. mansoni (P = .70).Children of mothers with hookworm or  mothers  and 4. SM. perstans did not differ when stratified by maternal malaria status. An association between maternal S. mansoni and childhood parasitemia was observed only in the presence of maternal malaria  among children of mothers with hookworm and Additionally we observed an increased rate of childhood clinical malaria in children of mothers with malaria compared with children of mothers without malaria. Whereas other studies have described associations between placental malaria infection and childhood malaria , 42, 43,M. perstans infections were at increased risk of peripheral malaria parasitemia [M. perstans and childhood malaria. This suggests that the association between maternal hookworm and childhood malaria may be independent of the association with maternal malaria and the other helminth infections.At enrolment, pregnant women with hookworm and asitemia , suggestM. perstans and childhood malaria persisted after adjusting for location of residence. However, we were unable to accurately assess the contribution of malaria transmission in the observed associations because we did not measure malaria exposure at the household or individual level. In the multivariable analyses, we adjusted for geographical location of residence assuming homogenous malaria transmission zones within an area of 4 km in diameter, which may not be sensitive to within-area transmission variations, and therefore we cannot exclude a role for malaria transmission in the observed associations.A possible explanation for these findings is that mothers with helminths and malaria in pregnancy came from a high malaria transmission environment and that the child's increased malaria risk was partly due to this. The risk of maternal malaria varied significantly by geographical zone of residence , suggestS. mansoni coinfection [S. mansoni coinfection, but we observed that children of S. mansoni–infected mothers were at higher risk of malaria parasitemia only if the mother also had malaria. This result should be interpreted with caution because it is not consistent with the association between clinical malaria and maternal S. mansoni, and we cannot rule out that this finding was due to chance alone. However, the result suggests the hypothesis that exposure to both malaria and helminths is required to alter the way the fetus's initial response to malaria is primed. In fact this may also be the case for the malaria–hookworm interaction. Although the associations between maternal hookworm and childhood malaria were statistically similar between children of mothers with and without malaria, the point estimates were lower in the strata of mothers without malaria. There may have been some misclassification of maternal malaria and, if exposure of the fetus to both malaria and hookworm is required for the observed effects, the observed trend in hazard ratios could have occurred if about 20% of the “no malaria” mothers actually had malaria at some time during the pregnancy. This would explain the absence of interaction between maternal malaria and hookworm or M. perstans.Previous studies have reported greater malaria morbidity associated with nfection . In our M. perstans infection might be more susceptible to malaria infection. However, adjusting for maternal malaria did not alter the associations between maternal hookworm or M. perstans infections and childhood malaria, suggesting that these helminth infections are not simply a marker for genetic susceptibility to malaria.A possible immunological explanation for the observed results is that fetal exposure to maternal helminth and malaria infections may induce T-cell hyporesponsiveness, downmodulating immunity to helminths  and malaS. mansoni infections [We used data collected from the EMaBS, a trial that investigated whether anthelminthic treatment during pregnancy could alter the effects of prenatal helminth exposure. Anthelminthic treatment in pregnancy was effective , but albfections . This cofections .Our major limitation was the use of single samples for ascertainment of maternal malaria and helminth infections. Although microscopy is the gold standard in malaria diagnosis, sensitivity is low in pregnancy due to low parasite densities and placental sequestration. In a review , the pooThis study provides the first report of an association between helminth infections in pregnancy and malaria in the offspring and suggests that helminth infections in pregnancy may increase the overall burden of childhood malaria in regions of coendemicity. The association between maternal hookworm and childhood malaria was consistent for clinical malaria episodes and asymptomatic parasitemia. However, the mechanism is unclear, and studies are needed to elucidate the significance of maternal helminth infections on fetal and early childhood antimalarial responses. Our findings support the strategy of integrated malaria–helminth control to accelerate the reduction of malaria morbidity and provide pertinent knowledge for the evaluation of malaria vaccine trials because results might be modified by concurrent helminth infections in pregnancy.Supplementary materials are available at The Journal of Infectious Diseases online . Supplementary materials consist of data provided by the author that are published to benefit the reader. The posted materials are not copyedited. The contents of all supplementary data are the sole responsibility of the authors. Questions or messages regarding errors should be addressed to the author.