Over the last decades, malaria has declined substantially in The Gambia but its transmission has not been interrupted. In order to better target control interventions, it is essential to understand the dynamics of residual transmission. This prospective cohort study was conducted between June 2013 and April 2014 in six pairs of villages across The Gambia. Blood samples were collected monthly during the transmission season (June-December) from all residents aged ≥6 months (4,194 individuals) and then in April (dry season). Entomological data were collected monthly throughout the malaria transmission season. Ownership of Long-Lasting Insecticidal Nets was 71.5% (2766/3869). Incidence of malaria infection and clinical disease varied significantly across the country, with the highest values in eastern (1.7/PYAR) than in central (0.2 /PYAR) and western (0.1/PYAR) Gambia. Malaria infection at the beginning of the transmission season was significantly higher in individuals who slept outdoors (HR = 1.51, 95% CI: 1.02–2.23, p = 0.04) and in those who had travelled outside the village (HR = 2.47, 95% CI: 1.83–3.34, p <0.01). Sub-patent infections were more common in older children (HR = 1.35, 95% CI: 1.04–1.6, p <0.01) and adults (HR = 1.53, 95% CI: 1.23–1.89, p<0.01) than in younger children. The risk of clinical malaria was significantly higher in households with at least one infected individual at the beginning of the transmission season (HR = 1.76, p<0.01). Vector parity was significantly higher in the eastern part of the country, both in the south (90.7%, 117/129, p<0.01) and the north bank (81.1%, 227/280, p<0.01), than in the western region (41.2%, 341/826), indicating higher vector survival. There is still significant residual malaria transmission across The Gambia, particularly in the eastern region. Additional interventions able to target vectors escaping Long-Lasting Insecticidal Nets and indoor residual spraying are needed to achieve malaria elimination.

BackgroundAs indicators of burden of malaria have substantially decreased in The Gambia, reaching a pre-elimination status may be attainable. Achieving this goal requires in-depth understanding of the current burden of Plasmodium falciparum infection.MethodsA nationwide cross-sectional survey was conducted in 2012 to determine the prevalence of P.falciparum infection, and to describe its heterogeneity and associated risk factors. Finger-prick blood samples were collected for microscopy, species-specific PCR and haemoglobin measurement.ResultsA total of 9,094 participants were included and median age was 11.9 years (IQR 5, 28). Overall prevalence of P. falciparum was 16.01 % with marked heterogeneity between sites (4.32–36.75 %) and within villages in each site (1.63–49.13 %). Across all sites, 51.17 % (745/1,456) of infections were asymptomatic and 35.61 % (448/1,258) were sub-microscopic. The odds of P. falciparum infection were higher in older children; 5–15 years (OR = 1.90; 95 % CI 1.60–2.26), adults (OR = 1.48; 95 % CI 1.24–1.78) and participants with moderate anaemia (OR = 1.62; 95 % CI 1.32–1.99).ConclusionsThe current malaria control interventions are not sufficient to interrupt transmission in The Gambia as malaria prevalence is still relatively high in the eastern part of the country. New interventions aiming at interrupting transmission are needed and should be urgently evaluated.

Cultured human pathogens may differ significantly from source populations. To investigate the genetic basis of laboratory adaptation in malaria parasites, clinical Plasmodium falciparum isolates were sampled from patients and cultured in vitro for up to three months. Genome sequence analysis was performed on multiple culture time point samples from six monoclonal isolates, and single nucleotide polymorphism (SNP) variants emerging over time were detected. Out of a total of five positively selected SNPs, four represented nonsense mutations resulting in stop codons, three of these in a single ApiAP2 transcription factor gene, and one in SRPK1. To survey further for nonsense mutants associated with culture, genome sequences of eleven long-term laboratory-adapted parasite strains were examined, revealing four independently acquired nonsense mutations in two other ApiAP2 genes, and five in Epac. No mutants of these genes exist in a large database of parasite sequences from uncultured clinical samples. This implicates putative master regulator genes in which multiple independent stop codon mutations have convergently led to culture adaptation, affecting most laboratory lines of P. falciparum. Understanding the adaptive processes should guide development of experimental models, which could include targeted gene disruption to adapt fastidious malaria parasite species to culture.

BackgroundIron deficiency causes long-term adverse consequences for children and is the most common nutritional deficiency worldwide. Observational studies suggest that iron deficiency anemia protects against Plasmodium falciparum malaria and several intervention trials have indicated that iron supplementation increases malaria risk through unknown mechanism(s). This poses a major challenge for health policy. We investigated how anemia inhibits blood stage malaria infection and how iron supplementation abrogates this protection.MethodsThis observational cohort study occurred in a malaria-endemic region where sickle-cell trait is also common. We studied fresh RBCs from anemic children (135 children; age 6–24 months; hemoglobin < 11 g/dl) participating in an iron supplementation trial (ISRCTN registry, number ISRCTN07210906) in which they received iron (12 mg/day) as part of a micronutrient powder for 84 days. Children donated RBCs at baseline, Day 49, and Day 84 for use in flow cytometry-based in vitro growth and invasion assays with P. falciparum laboratory and field strains. In vitro parasite growth in subject RBCs was the primary endpoint.FindingsAnemia substantially reduced the invasion and growth of both laboratory and field strains of P. falciparum in vitro (~ 10% growth reduction per standard deviation shift in hemoglobin). The population level impact against erythrocytic stage malaria was 15.9% from anemia compared to 3.5% for sickle-cell trait. Parasite growth was 2.4 fold higher after 49 days of iron supplementation relative to baseline (p < 0.001), paralleling increases in erythropoiesis.InterpretationThese results confirm and quantify a plausible mechanism by which anemia protects African children against falciparum malaria, an effect that is substantially greater than the protection offered by sickle-cell trait. Iron supplementation completely reversed the observed protection and hence should be accompanied by malaria prophylaxis. Lower hemoglobin levels typically seen in populations of African descent may reflect past genetic selection by malaria.FundingNational Institute of Child Health and Development, Bill and Melinda Gates Foundation, UK Medical Research Council (MRC) and Department for International Development (DFID) under the MRC/DFID Concordat.

BackgroundMalaria morbidity and mortality has declined in recent years in a number of settings. The ability to describe changes in malaria transmission associated with these declines is important in terms of assessing the potential effects of control interventions, and for monitoring and evaluation purposes.MethodsData from five cross-sectional surveys conducted in Farafenni and surrounding villages on the north bank of River Gambia between 1988 and 2011 were compiled. Antibody responses to MSP-119 were measured in samples from all surveys, data were normalized and expressed as seroprevalence and seroconversion rates (SCR) using different mathematical models.ResultsResults showed declines in serological metrics with seroprevalence in children aged one to 5 years dropping from 19 % (95 % CI 15–23 %) in 1988 to 1 % (0–2 %) in 2011 (p value for trend in proportions < 0.001) and the SCR dropping from 0.069 year−1 (0.059–0.080) to 0.022 year−1 (0.017–0.028; p = 0.004). The serological data were consistent with previously described drops in both parasite prevalence in children aged 1–5 years (62 %, 57–66 %, in 1988 to 2 %, 0–4 %, in 2011; p < 0.001), and all-cause under five mortality rates (37 per 1000 person-years, 34–41, in 1990 to 17, 15–19, in 2006; p = 0.059).ConclusionsThis analysis shows accurate reconstruction of historical malaria transmission patterns in the Farafenni area using anti-malarial antibody responses. Demonstrating congruence between serological measures, and conventional clinical and parasitological measures suggests broader utility for serology in monitoring and evaluation of malaria transmission.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0939-1) contains supplementary material, which is available to authorized users.

Background: Antibody responses have been used to characterise transmission and exposure history in malaria-endemic settings for over a decade. Such studies have typically been conducted on well-standardised enzyme-linked immunosorbent assays (ELISAs). However, recently developed quantitative suspension array technologies (qSAT) are now capable of high-throughput and multiplexed screening of up to hundreds of analytes at a time. This study presents a customised protocol for the Luminex MAGPIX© qSAT using a diverse set of malaria antigens. The aim is to develop a standardised assay for routine serological surveillance that is implementable across laboratories and epidemiological settings. Methods: A panel of eight Plasmodium falciparum recombinant antigens, associated with long- and short-lived antibody responses, was designed for the Luminex MAGPIX© platform. The assay was optimised for key steps in the protocol: antigen-bead coupling concentration, buffer composition, serum sample dilution, and bead storage conditions. Quality control procedures and data normalisation methods were developed to address high-throughput assay processing.  Antigen-specific limits of quantification (LOQs) were also estimated using both in-house and WHO reference serum as positive controls. Results: Antigen-specific bead coupling was optimised across five serum dilutions and two positive controls, resulting in concentrations operational within stable analytical ranges. Coupled beads were stable after storage at room temperature (22⁰C) for up to eight weeks. High sensitivity and specificity for distinguishing positive and negative controls at serum sample dilutions of 1:500 (AUC 0.94 95%CI 0.91-0.96) and 1:1000 (AUC 0.96 95%CI 0.94-0.98) were observed. LOQs were also successfully estimated for all analytes but varied by antigen and positive control. Conclusions: This study demonstrates that developing a standardised malaria-specific qSAT protocol for a diverse set of antigens is achievable, though further optimisations may be required. Quality control and data standardisation methods may also be useful for future analysis of large sero-epidemiological surveys.

Background. Many genes of the malaria parasite Plasmodium falciparum show clonally variant expression regulated at the epigenetic level. These genes participate in fundamental host-parasite interactions and contribute to adaptive processes. However, little is known about their expression patterns during human infections. A peculiar case of clonally variant genes are the 2 nearly identical clag3 genes, clag3.1 and clag3.2, which mediate nutrient uptake and are linked to resistance to some toxic compounds.Methods. We developed a procedure to characterize the expression of clag3 genes in naturally infected patients and in experimentally infected human volunteers.Results. We provide the first description of clag3 expression during human infections, which revealed mutually exclusive expression and identified the gene predominantly expressed. Adaptation to culture conditions or selection with a toxic compound resulted in isolate-dependent changes in clag3 expression. We also found that clag3 expression patterns were reset during transmission stages.Conclusions. Different environment conditions select for parasites with different clag3 expression patterns, implying functional differences between the proteins encoded. The epigenetic memory is likely erased before parasites start infection of a new human host. Altogether, our findings support the idea that clonally variant genes facilitate the adaptation of parasite populations to changing conditions through bet-hedging strategies.

BackgroundPrimaquine (PQ) actively clears mature Plasmodium falciparum gametocytes but in glucose-6-phosphate dehydrogenase deficient (G6PDd) individuals can cause hemolysis. We assessed the safety of low-dose PQ in combination with artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DP) in G6PDd African males with asymptomatic P. falciparum malaria.Methods and findingsIn Burkina Faso, G6PDd adult males were randomized to treatment with AL alone (n = 10) or with PQ at 0.25 (n = 20) or 0.40 mg/kg (n = 20) dosage; G6PD-normal males received AL plus 0.25 (n = 10) or 0.40 mg/kg (n = 10) PQ. In The Gambia, G6PDd adult males and boys received DP alone (n = 10) or with 0.25 mg/kg PQ (n = 20); G6PD-normal males received DP plus 0.25 (n = 10) or 0.40 mg/kg (n = 10) PQ. The primary study endpoint was change in hemoglobin concentration during the 28-day follow-up. Cytochrome P-450 isoenzyme 2D6 (CYP2D6) metabolizer status, gametocyte carriage, haptoglobin, lactate dehydrogenase levels and reticulocyte counts were also determined.In Burkina Faso, the mean maximum absolute change in hemoglobin was -2.13 g/dL (95% confidence interval [CI], -2.78, -1.49) in G6PDd individuals randomized to 0.25 PQ mg/kg and -2.29 g/dL (95% CI, -2.79, -1.79) in those receiving 0.40 PQ mg/kg. In The Gambia, the mean maximum absolute change in hemoglobin concentration was -1.83 g/dL (95% CI, -2.19, -1.47) in G6PDd individuals receiving 0.25 PQ mg/kg. After adjustment for baseline concentrations, hemoglobin reductions in G6PDd individuals in Burkina Faso were more pronounced compared to those in G6PD-normal individuals receiving the same PQ doses (P = 0.062 and P = 0.022, respectively). Hemoglobin levels normalized during follow-up. Abnormal haptoglobin and lactate dehydrogenase levels provided additional evidence of mild transient hemolysis post-PQ.ConclusionsSingle low-dose PQ in combination with AL and DP was associated with mild and transient reductions in hemoglobin. None of the study participants developed moderate or severe anemia; there were no severe adverse events. This indicates that single low-dose PQ is safe in G6PDd African males when used with artemisinin-based combination therapy.Trial registrationClinicaltrials.gov NCT02174900Clinicaltrials.gov NCT02654730