Search

Genetic diversity of surface exposed and stage specific Plasmodium falciparum immunogenic proteins pose a major roadblock to developing an effective malaria vaccine with broad and long-lasting immunity. We conducted a prospective genetic analysis of candidate antigens (msp1, ama1, rh5, eba175, glurp, celtos, csp, lsa3, Pfsea, trap, conserved chrom3, hyp9, hyp10, phistb, surfin8.2, and surfin14.1) for malaria vaccine development on 2375 P. falciparum sequences from 16 African countries. We described signatures of balancing selection inferred from positive values of Tajima’s D for all antigens across all populations except for glurp. This could be as a result of immune selection on these antigens as positive Tajima’s D values mapped to regions with putative immune epitopes. A less diverse phistb antigen was characterised with a transmembrane domain, glycophosphatidyl anchors between the N and C- terminals, and surface epitopes that could be targets of immune recognition. This study demonstrates the value of population genetic and immunoinformatic analysis for identifying and characterising new putative vaccine candidates towards improving strain transcending immunity, and vaccine efficacy across all endemic populations.

Background This cross-sectional study was carried out in different settlements of Gabon to determine the influence of single or multiple parasite carriage on haemoglobin (Hb) levels. Methods Between April 2015 and June 2016, healthy volunteers from urban, peri-urban and rural areas were screened for malaria, blood filariasis and intestinal parasitic infections using microscopic methods. Hb concentration was measured with a Hemocue analyser. The association between parasite carriage and anaemia was assessed. Results Among the 775 volunteers examined, 319 (41.2%) were from rural villages and 76.0% were adults. Filariasis, intestinal parasitic infections, Plasmodium falciparum and polyparasitism were detected in 15.6, 14.6, 9.5 and 6.8% of participants, respectively. Anaemia prevalence was 72.6%, with rates of mild, moderate and severe anaemia being 30.9, 61.1 and 8.0%, respectively. The median Hb level was lowest in the presence of hookworms (7.1 g/dl [interquartile range {IQR} 6.8–7.5]), Schistosoma intercalatum (6.9 g/dl), Trichuris trichiura (10.1 g/dl [IQR 8.9–11.5]) and Plasmodium falciparum (10.0 g/dl [IQR 9.1–11.2]) compared with filariaemia (12.1 g/dl [IQR 10.5–13.2]) (p=0.03). Moderate to severe anaemia predominated among those single-infected with P. falciparum (69.5%) or co-infected with intestinal parasitic infections and P. falciparum (76.2%), while it was found in only 23.2% of individuals with filariasis. All participants with soil-transmitted helminths and more than half with a Blastocystis sp. (68.8%) infection had moderate anaemia. Conclusions The prevalence of anaemia is high. Asymptomatic parasite carriage is associated with anaemia in this surveyed population in Gabon.

Background: To perform molecular epidemiologic studies based on large cohorts, material such as RDTs or filter papers are useful for biological sample collection and extraction of RNA or DNA of good quality. Thus, we aimed to assess the quality of DNA extracted from malaria rapid diagnostic tests (RDTs) stored at various temperatures for the analysis of Plasmodium falciparum genetic diversity. Methods: Febrile patients benefitted from free malaria diagnosis using microscopy in a malaria sentinel site, at the Regional Hospital Estuaire-Melen, in Gabon, in 2015. P. falciparum isolates were collected onto one filter paper and 2 similar RDTs devices (Acon®) per patient. Nucleic acids were extracted with QiAmp Qiagen kit from paper and RDTs and the quality of the DNA was analyzed by msp1 gene amplification. Results: Msp1gene amplification was achieved in nucleic acids extracted from all filter papers and RDTs devices (n = 45, 100%). K1 alleles were detected in 93.3% (n = 42/45) of the samples and Mad20 alleles in 73.3% (n = 33/45). The number and the intensity of K1 and/or Mad20 fragments were comparable according to the sample collection material and the storage conditions (room temperature vs -20°C) of the samples. The size of the fragments indicating allelic diversity was comparable in 80% (n=36) of the samples. Conclusion: These data show that RDTs are a valuable source of DNA for malaria parasite genetic polymorphism analysis. Storage conditions of the devices did not influence the quality of DNA extracted from RDTs device, although some alleles may be missed.

Background For uncomplicated Plasmodium falciparum malaria, highly efficacious single-dose treatments are expected to increase compliance and improve treatment outcomes, and thereby may slow the development of resistance. The efficacy and safety of a single-dose combination of artefenomel (800 mg) plus ferroquine (400/600/900/1200 mg doses) for the treatment of uncomplicated P. falciparum malaria were evaluated in Africa (focusing on children ≤ 5 years) and Asia. Methods The study was a randomized, double-blind, single-dose, multi-arm clinical trial in patients aged > 6 months to kelch-13 mutations were explored. Results A total of 373 patients were treated: 289 African patients ≤ 5 years (77.5%), 64 African patients > 5 years and 20 Asian patients. None of the treatment arms met the target efficacy criterion for PCR-adjusted ACPR at Day 28 (lower limit of 95% confidence interval [CI] > 90%). PCR-adjusted ACPR at Day 28 [95% CI] in the PP Set ranged from 78.4% [64.7; 88.7%] to 91.7% [81.6; 97.2%] for the 400 mg to 1200 mg ferroquine dose. Efficacy rates were low in Vietnamese patients, ranging from 20 to 40%. A clear relationship was found between drug exposure (artefenomel and ferroquine concentrations at Day 7) and efficacy (primary endpoint), with higher concentrations of both drugs resulting in higher efficacy. Six distinct kelch-13 mutations were detected in parasite isolates from 10/272 African patients (with 2 mutations known to be associated with artemisinin resistance) and 18/20 Asian patients (all C580Y mutation). Vomiting within 6 h of initial artefenomel administration was common (24.6%) and associated with lower drug exposures. Conclusion The efficacy of artefenomel/ferroquine combination was suboptimal in African children aged ≤ 5 years, the population of interest, and vomiting most likely had a negative impact on efficacy. Trial registration ClinicalTrials.gov, NCT02497612. Registered 14 Jul 2015, https://clinicaltrials.gov/ct2/show/NCT02497612?term=NCT02497612&draw=2&rank=1

Purpose Artesunate–amodiaquine (AS–AQ) and artemether–lumefantrine (AL) have been widely used for the treatment of uncomplicated Plasmodium falciparum malaria since 2005 in Gabon. Since 2011, a rebound of malaria morbidity has been observed in this country, while no survey evaluating ACT efficacy was performed. During the same period, parasite resistance against artemisinin has been reported in Asia. The aim of this study was to assess the efficacy and tolerability of these two drugs in two sentinel sites of Gabon 10 years after their implementation. Methods Children aged from 12 to 144 months with uncomplicated malaria were recruited at the Regional Hospital of Melen, Libreville and in the Urban Health Center of Franceville between March 2014 and September 2015. The therapeutic efficacy was evaluated according to the WHO 2008 protocol of 28-day follow-up and PCR-uncorrected/corrected treatment outcomes were assessed. Results One hundred and eighty-five children (98 ASAQ and 89 AL) were followed up until day 28. The PCR-corrected ACPR was 98.9% for AS–AQ and 96.4% for AL. Late therapeutic failure rate was 3.6% and 1.1% for AL and AS–AQ, respectively (p = 0.2). Adverse events and serious adverse events were rarely observed with both treatments. Conclusion AS–AQ and AL are still efficacious and well-tolerated for the treatment of uncomplicated malaria in Gabonese children.

Ebb and flow of parasite populations The population genetics of the malaria parasite Plasmodium falciparum across Africa is poorly understood but important to know for grasping the risks and dynamics of the spread of drug resistance. Harnessing the power of genomics, Amambua-Ngwa et al. of the Plasmodium Diversity Network Africa found substantial population structure within Africa that is consistent with human and vector population divergence (see the Perspective by Sibley). Specific signatures of selection by antimalarial drugs were detected, along with indications of the effect of colonization and slavery. Furthermore, whole-genome sequencing showed that there is extensive gene flow among the different regions and that Ethiopia has a distinctive population of P. falciparum , which may be indicative of coexistence with another malaria parasite, P. vivax. Science , this issue p. 813 ; see also p. 752

Summary Background Artemisinin-based combination therapies (ACTs) are the recommended first-line treatment for uncomplicated Plasmodium falciparum malaria. Ferroquine is a new combination partner for fast-acting ACTs such as artesunate. We aimed to assess different doses of ferroquine in combination with artesunate against uncomplicated P falciparum malaria in a heterogeneous population in Africa. Methods We did a phase 2, multicentre, parallel-group, double-blind, randomised, dose-ranging non-inferiority trial at eight African hospitals (two in Gabon, three in Burkina Faso, one in Benin, and two in Kenya). We recruited patients presenting with acute P falciparum monoinfection (1000–200 000 parasites per μL), and a central body temperature of at least 37·5°C or history of fever in the past 24 h. We assessed patients in two sequential cohorts: cohort 1 contained adults (bodyweight >50 kg) and adolescents (aged ≥14 years, >30 kg), and cohort 2 contained children (aged 2–13 years, 15–30 kg). We randomly assigned patients (1:1:1:1) to receive artesunate 4 mg/kg per day plus ferroquine 2 mg/kg, 4 mg/kg, or 6 mg/kg, given double-blind once per day for 3 days, or ferroquine monotherapy 4 mg/kg per day given single-blind (ie, allocation was only masked from the patient) once per day for 3 days. We did 14 patient visits (screening, 3 treatment days and 48 h post-treatment surveillance, a visit on day 7, then one follow-up visit per week until day 63). The primary endpoint was non-inferiority of treatment in terms of PCR-corrected cure rate against a reference value of 90%, with a 10% non-inferiority margin, assessed in patients treated without major protocol deviations for parasitologically confirmed malaria. We assessed safety in all treated patients. This study is registered with ClinicalTrials.gov, number NCT00988507, and is closed. Findings Between Oct 16, 2009, and Sept 22, 2010, we randomly assigned 326 eligible patients to treatment groups, with last follow-up visit on Dec 1, 2010. 284 patients (87%) were available for per-protocol analyses. At day 28, PCR-confirmed cure was noted in 68 (97%, 95% CI 90–100) of 70 patients treated with ferroquine 2 mg/kg plus artesunate, 73 (99%, 93–100) of 74 with ferroquine 4 mg/kg plus artesunate, 71 (99%, 93–100) of 72 with ferroquine 6 mg/kg plus artesunate, and 54 (79%, 68–88) of 68 with ferroquine 4 mg/kg monotherapy. The three dose groups of ferroquine plus artesunate met the non-inferiority hypothesis. The most common adverse events were headache in cohort 1 (30 [19%] of 162 patients) and worsening malaria in cohort 2 (23 [14%] of 164 patients); occurrences were similar between treatment groups. Interpretation Ferroquine combined with artesunate was associated with high cure rates and was safe at all doses tested, and could be a promising new drug combination for the treatment of P falciparum malaria. Ferroquine could also partner other drugs to establish a new generation of antimalarial combinations, especially in regions that have developed resistance to ACTs. Funding Sanofi.

The present study determined and compared the genetic diversity of Plasmodium falciparum strains infecting children living in 2 areas from Gabon with different malaria endemicity. Blood samples were collected from febrile children from 2008 to 2009 in 2 health centres from rural (Oyem) and urban (Owendo) areas. Genetic diversity was determined in P. falciparum isolates by analyzing the merozoite surface protein-1 (msp1) gene polymorphism using nested-PCR. Overall, 168 children with mild falciparum malaria were included. K1, Ro33, and Mad20 alleles were found in 110 (65.5%), 94 (55.9%), and 35 (20.8%) isolates, respectively, without difference according to the site (P>0.05). Allelic families’ frequencies were comparable between children less than 5 years old from the 2 sites; while among the older children the proportions of Ro33 and Mad20 alleles were 1.7 to 2.0 fold higher at Oyem. Thirty-three different alleles were detected, 16 (48.5%) were common to both sites, and 10 out of the 17 specific alleles were found at Oyem. Furthermore, multiple infection carriers were frequent at Oyem (57.7% vs 42.2% at Owendo; P=0.04) where the complexity of infection was of 1.88 (±0.95) higher compared to that found at Owendo (1.55±0.75). Extended genetic diversity of P. falciparum strains infecting Gabonese symptomatic children and high multiplicity of infections were observed in rural area. Alleles common to the 2 sites were frequent; the site-specific alleles predominated in the rural area. Such distribution of the alleles should be taken into accounts when designing MSP1 or MSP2 malaria vaccine.

Plasmodium population dynamics analysis may help to assess the impact of malaria control strategies deployment. In Gabon, new strategies have been introduced, but malaria is still a public health problem marked by a rebound of the prevalence in 2011. The aim of the study was to investigate the spatial and temporal distribution of P. falciparum strains in different areas in Gabon during a period of malaria transmission transition, between 2008 and 2011. A total of 109P. falciparum isolates were genotyped using nested-PCR of Pfmsp1 and Pfmsp2 genes. 3D7, FC27 and K1 allele frequencies were comparable between sites (p=0.9); those of Ro33 (93.6%; 44/47) and Mad20 (60%; 12/20) were significantly higher in isolates from Oyem (p0.01) and Port-Gentil (p=0.02), respectively. The frequency of multiples infections (77%) and the complexity of infection (2.66±1.44) were the highest at Oyem. Pfmsp1 gene analysis highlighted a trend of a decreasing frequency of K1 family, in Libreville and Oyem between 2008 and 2011; while that of Ro33 (p0.01) and Mad20 (p0.01) increased. The prevalence of multiple infections was comparable between both periods in each site: 42.2% vs 47.6% (p=0.6) in Libreville and 57.7% vs 61.7% in Oyem (p=0.8). In contrast, in 2011, the COI tends to be higher in Libreville and did not vary in Oyem. These data confirm an extended genetic diversity of P. falciparum isolates over time and according to geographic location in Gabon. Nevertheless, the impact of the deployment of malaria control strategies on the parasites genetic profile is not clearly established here.

Changes of lipid parameter concentrations are observed in patients suffering from malaria. However, there are few data on their evolution during the course of the infection and their relationship with the infection parameters. The levels of triglycerides (TG) and total cholesterol (TC) were measured during and after uncomplicated Plasmodium falciparum malaria in 122 children. The relationship with the antimalarial drug treatment, the parasite density and haemoglobin levels was also assessed. Mean TG levels were high before the treatment (1.9 ± 1.2 mmol/L) and at day 3 post-treatment (2.3 ± 1.3 mmol/L), then subsequently decreased to reach normal level from day 7. A negative correlation was found between haemoglobin (Hb) levels and TG (rho = -0.47; p < 0.01); previous high parasitaemia at inclusion was associated with a subsequent increase of TG levels after treatment administration. The majority of patients with hypertriglyceridemia were anaemic at day 3 (n = 20/20) and at day 7 (n = 18/19). Only 5 (4.1%) patients had hypertriglyceridemia at day 28. A trend toward a negative correlation between TC levels and parasite density was observed (rho = -0.18; p = 0.05). Mean TC concentration was significantly lower at day 0 when all the patients were parasitaemic (3.0 ± 1.0 mmol/L) compared to day 3 (3.7 ± 1.2 mmol/L) (p = 0.01). Mean CT was low during the first week and at day 28, 71 (58.2%) children still had a subnormal CT levels. No relationship was found with the type of antimalarial drug. Transient hypertriglyceridemia and hypocholesterolemia are observed during the course of P. falciparum infection. High TG levels seem to be related to the malaria related haemolysis. Key words: Malaria, cholesterol, triglycerides, antimalarial, Gabon.

A total of 415 samples from febrile children exhibiting either concordant (n = 108) or discordant (n = 307) results between microscopy, the gold standard diagnostic test, and two Rapid Diagnostic Tests (RDTs), OptiMAL-IT?(pLDH) and Acon?HRP2, were analysed using polymerase chain reaction (PCR) from May to December 2011 in Gabon. The aim of the study was to analyse these discrepancies using poly-merase chain reaction (PCR). Nested PCR targeting the Plasmodium ssrRNA gene was used to distinguish P. falciparum, P. malariae and P. ovale. Plasmodium falciparum was the only malaria species identified. Discrepancies frequently involved samples that were negative by microscopy and positive by Acon?HRP2 (90%) or Optimal-it?(86%). The PCR assay detected submicroscopic infection in almost 23% of the microscopy-negative samples, whereas plasmodial DNA was not found in 77% of the Acon?HRP2 positive-microscopy negative samples. Although results obtained with Optimal-it? were more frequently concordant with those of PCR genotyping, the low specificity of Optimal-iT?for non-falciparum malaria parasite detection resulted in a high proportion of false negative RDTs (90%) and a high frequency of tests with faint line intensity. The present study highlights the specific attributes of the different methods used to identify malaria parasite below the microscopy level of detection. RDT results that were discordant with either microscopy or PCR as the gold standard could represent a challenge for rapid, accurate fever case management in malaria endemic areas. It is necessary to pursue the development of more precise and more sensitive point-of-care diagnostic tools for malaria.

BACKGROUND Six years after the implementation of intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) in Gabon, its impact on placental malaria and pregnancy outcomes remains unknown. METHODS Age, gestational data, use of IPTp-SP and birth weight were recorded during a hospital-based cross-sectional survey performed in 2011 in 387 women at the end of pregnancy. RESULTS Malaria prevalence was 6.7 and 5.3% in peripheral and placental blood respectively. Overall, 59.0% women took at least two IPTp-SP doses which was associated with 50% reduction of Plasmodium; (P.) falciparum infection in primigravidae. Previous malaria treatment was a risk factor for peripheral P. falciparum infection, while uptake of IPTp-SP was associated with reduced parasitaemia. Anaemia prevalence was 38.0%, low birth weight and prematurity rates were 6.0 and 12.0% respectively. Young age was associated with a higher frequency of malaria, anaemia, low birth weight and preterm delivery (p

The frequency of dhfr and dhps point mutations was assessed in Plasmodium falciparum isolates from pregnant women in Libreville.PCR-restriction fragment length polymorphism of polymorphic codons of the dhfr gene (51, 59 and 108) and the dhps gene (436, 437 and 540) was performed in matched peripheral and placental blood samples.The proportion of multiple mutations was high (98%), and was not different between women with and without a history of intermittent preventive treatment with sulfadoxine/pyrimethamine (IPTp/SP). The prevalence of triple dhfr mutation was 80%, and that of quadruple and quintuple mutations was 53% and 22%, respectively. The Glu540 mutation was present in two isolates. The concordance of resistant alleles in matched peripheral and placental isolates was90% for both genes.These findings underline the need for a regular assessment of the relationship between the presence of resistant isolates and in vitro/in vivo IPTp/SP efficacy, and evaluation of an alternative drug.

Introduction. The characterization of genetic profile ofPlasmodiumisolates from different areas could help in better strategies for malaria elimination. This study aimed to compareP. falciparumdiversity in two African countries.Methods. Isolates collected from 100 and 73falciparummalaria infections in sites of Côte d’Ivoire (West Africa) and Gabon (Central Africa), respectively, were analyzed by a nested PCR amplification ofmsp1 andmsp2 genes.Results. The K1 allelic family was widespread in Côte d’Ivoire (64.6%) and in Gabon (56.6%). Formsp2, the 3D7 alleles were more prevalent (>70% in both countries) compared to FC27 alleles. In Côte d’Ivoire, the frequencies of multiple infections withmsp1 (45.1%) andmsp2 (40.3%) were higher than those found for isolates from Gabon, that is, 30.2% withmsp1 and 31.4% withmsp2. The overall complexity of infection was 1.66 (SD = 0.79) in Côte d’Ivoire and 1.58 (SD = 0.83) in Gabon. It decreased with age in Côte d’Ivoire in contrast to Gabon.Conclusion. Differences observed in some allelic families and in complexity profile may suggest an impact of epidemiological facies as well as immunological response on genetic variability ofP. falciparum.

We assessed Plasmodium (P.) falciparum allelic diversity based on clinical severity and age. The study was conducted from 2011 to 2012 in Libreville, Gabon where malaria prevalence was 24.5%. The polymorphism of the merozoite surface protein-1 (msp1) locus was analyzed in isolates from patients with complicated and uncomplicated malaria. Blood was collected on filter paper. After DNA extraction, genotyping of the msp1 gene was performed using nested PCR. The K1, Ro33, and Mad20 allelic families were detected in 71 (63%), 64 (57%), and 38 (34%) of the 112 analyzed samples, respectively. Overall, 17 K1 and 11 Mad20 alleles were detected. There was no association between msp1 allelic families and age. Mad20 allelic diversity increased with the severity of malaria. The number of K1 and Mad20 alleles decreased with age. The multiplicity of infection (MOI) was 1–6 genotypes and the complexity of infection (COI) 1.8 ± 1. The COI differed based on age: it was 1.9 (±1.1) in the isolates from adults, 1.8 (±1.1) in those from 0–5 year-old children, whereas it tended to be lower (1.6 ± 0.8) in those from 6–15 year-old children. Extensive genetic diversity is found in P. falciparum strains circulating in Libreville. The number of specific msp1 alleles increased with clinical severity, suggesting an association between the diversity and the severity of malaria.

Item does not contain fulltext We measured natural killer (NK) cell cytotoxicity and cortisol and prolactin concentrations in peripheral venous blood samples obtained from pregnant Gabonese women at the time of delivery. The NK cell-mediated cytotoxicity against Plasmodium falciparum-infected erythrocytes in vitro was lower in samples obtained from primiparous women than in samples obtained from multiparous women; cortisol concentrations were significantly higher in primiparous women than in multiparous women, and prolactin concentrations were significantly lower. The highest cortisol concentrations were found in the plasma of P. falciparum-infected primiparous women. A positive correlation was found between cortisol concentration and parasite load; an inverse correlation was found between the magnitude of the NK cell cytolytic effect and cortisol production. A positive correlation was found between this effect and prolactin production. Thus, depressed NK cell cytotoxicity against P. falciparum-infected erythrocytes is correlated with high cortisol concentrations and may contribute to increased susceptibility to malaria during pregnancy.

This study analyzed the relationship between intermittent preventive treatment with sulfadoxine-pyrimethamine (SP) (IPTp-SP), the rate of multiple resistant parasites and of submicroscopic gametocyte carriage among pregnant women at the beginning of IPTp implementation in Gabon (2005) and six years after (2011). The detection of pfdhfr and pfdhps gene mutations was performed by PCR-RFLP in Plasmodium (P.) falciparum positive samples collected from pregnant women in 2005 and 2011. Gametocytes carriage was detected by Pfs25mRNA amplification using QT-NASBA. Data were analyzed according to the time of collection (study period) and IPTp-SP doses. The proportion of isolates with at least a triple Pfdhfr mutation (n = 39/42, 92.9% versus 100%, n = 78/78)) and of those isolates with the S108N/C59R/N51I/S436A/A437G multiple mutation (17.9% versus 75.6%) significantly increased between 2005 and 2011 (p

Mutations in the Plasmodium falciparum K13-propeller domain have recently been shown to be important determinants of artemisinin resistance in Southeast Asia. This study investigated the prevalence of K13-propeller polymorphisms across sub-Saharan Africa. A total of 1212 P. falciparum samples collected from 12 countries were sequenced. None of the K13-propeller mutations previously reported in Southeast Asia were found, but 22 unique mutations were detected, of which 7 were nonsynonymous. Allele frequencies ranged between 1% and 3%. Three mutations were observed in >1 country, and the A578S was present in parasites from 5 countries. This study provides the baseline prevalence of K13-propeller mutations in sub-Saharan Africa.

Following the observed increase of malaria prevalence among older children in Gabon, a descriptive observational study was carried out in 2012 to determine the prevalence of malaria in adults presenting with fever in two health centres of Libreville, the capital city of Gabon. Thick- and thin-blood smears for malaria diagnosis were performed in febrile individuals aged more than 15 years old. Age, use of bed nets, previous antimalarial drug treatment, clinical symptoms, chest radiography results, and available haemoglobin data were also recorded. Among the 304 patients screened, the global malaria frequency was of 42.1%(n=128/34).Plasmodium (P). falciparumwas the only species identified. The proportion of patients with a clinical malaria requiring parenteral treatment was 38.5%, whereas 47.5% of outpatients had uncomplicated malaria. According to WHO classification, 14 (19.7%) infected patients had severe malaria; neurological and respiratory symptoms tended to be more frequent in case ofP. falciparuminfection. Anaemia was found in 51.5% adults and none had severe anaemia. Almost half of adults consulting for fever in two health centres of the urban city of Libreville have malaria. The use of insecticide-treated bed nets, the screening, and the treatment of individuals withP. falciparummicroscopic and submicroscopic asymptomatic infection or clinical malaria should be emphasized to reduce the transmission.

Background Considering malaria prevalence declines in parts of sub-Saharan Africa, such as Gabon, identification of the human infectious reservoir is important for successful malaria control. Microscopic and sub-microscopic parasites contribute to malaria transmission. The aim of the present study was to evaluate the proportion of microscopic and sub-microscopic gametocyte carriers among febrile patients in two different areas of Gabon. Methods Samples from febrile children aged less than 11 years old were collected from February 2008 to January 2009 at two health centres of Gabon. Patients were screened for the presence of asexual Plasmodium falciparum parasites. Gametocyte carriage was determined by microscopy and QT-NASBA. Results Gametocytes were detected in 5.3% (n = 16/304) of children by microscopy compared to 45.7% (n = 139/304) by QT-Nasba. Sub-microscopic gametocyte carriage (ie microscopy negative and QT-Nasba positive) was found in 89.2% (n = 124/139) of patients. Among patients with microscopically detected trophozoites, the proportion of sub-microscopic gametocyte (SMG) carriers was 58.4% (n = 118/202) and 6% in samples from children with negative slides (p < 0.01). In Oyem, where malaria prevalence is three-fold higher than in Owendo, SMG carriage was more frequent (49.0% vs 32.6% in Owendo; p < 0.01). Conclusion Sub-microscopic gametocytaemia is common among Gabonese febrile children. They might strongly contribute to maintain malaria transmission. However, further analysis of sub-microscopic parasite carriage among asymptomatic individuals will be helpful to better characterize malaria transmission.

Resistance by Plasmodium falciparum to antimalarial drugs has strongly hampered strategies for malaria control and elimination. Therefore, one of the goals of the World Health Organisation's new malaria control strategies is the rational and appropriate use of antimalarial drugs and in particular of artemisinin-based combination therapy (ACTs), currently used for the treatment of uncomplicated malaria; in order to delay the appearance of drug-resistant parasites. The unprescribed use of antimalarial drugs (self medication and parental administration to children) is a key component in the development of antimalarial drug resistance and must be controlled among patients living in malaria-endemic areas. The aim of our study was to estimate the frequency of this parental administration among febrile children and to identify the specific drugs used. Data were collected in two studies evaluating the proportion of malaria cases and the performance of rapid drug tests among febrile children seen in 2008-2009 at 3 hospitals, one in a rural area, one in an urban area, and the third in a semi-urban area. This parental medication administration was found among 21.4% of the 2543 children included in the studies. It was most common at the rural hospital (29%), which is also where malaria prevalence was highest (39%). Of the 548 children "medicated", 421, that is, almost 80%, were not infected. The antimalarial drugs used most frequently were ACTs (43.8%) and quinine (12%). In Gabon, as in other sub-Saharan countries, use of antimalarial drugs before consultation is common and is an obstacle to malaria control. Therefore, improving the rational use of these drugs by the population requires active outreach to the community about the risks of unprescribed medication.

The African continent continues to bear the greatest burden of malaria and the greatest diversity of parasites, mosquito vectors, and human victims. The evolutionary plasticity of malaria parasites and their vectors is a major obstacle to eliminating the disease. Of current concern is the recently reported emergence of resistance to the front-line drug, artemisinin, in South-East Asia in Plasmodium falciparum , which calls for preemptive surveillance of the African parasite population for genetic markers of emerging drug resistance. Here we describe the Plasmodium Diversity Network Africa (PDNA), which has been established across 11 countries in sub-Saharan Africa to ensure that African scientists are enabled to work together and to play a key role in the global effort for tracking and responding to this public health threat.

The ability of natural killer (NK) cells to produce gamma interferon (IFN-gamma) after ex vivo stimulation with crude schizont lysate of Plasmodium falciparum was studied in uninfected and P. falciparum-infected pregnant Gabonese women segregated according to the gravidity at the time of delivery. This activity was measured in purified NK cells as well as in whole blood from the periphery and cord. Crude schizont lysate-stimulated NK cells from primiparous women produced significantly more IFN-gamma than those from multiparous women (P0.001). Women with malaria infection produced more IFN-gamma than negative women in peripheral blood (P0.001) indicating that immunological determinants regulating the susceptibility to malaria in pregnant women are parasite-specific. These findings reveal that NK cells are major source of IFN-gamma when exposed to P. falciparum antigens in vitro in absence of any other co-stimulant.

Background Improving the understanding of childhood malarial anaemia may help in the design of appropriate management strategies. Methods A prospective observational study over a two-year period to assess the burden of anaemia and its relationship to Plasmodium falciparum infection and age was conducted in 8,195 febrile Gabonese children. Results The proportion of children with anaemia was 83.6% (n = 6830), higher in children between the ages of six and 23 months. Those under three years old were more likely to develop moderate to severe anaemia (68%). The prevalence of malaria was 42.7% and P. falciparum infection was more frequent in children aged 36–47 months (54.5%). The proportion of anaemic children increased with parasite density (p < 0.01). Most of infected children were moderately to severely anaemic (69.5%, p < 0.01). Infants aged from one to 11 months had a higher risk of developing severe malarial anaemia. In children over six years of age, anaemia occurrence was high (>60%), but was unrelated to P. falciparum parasitaemia. Conclusion Malaria is one of the main risk factors for childhood anaemia which represents a public health problem in Gabon. The risk of severe malarial anaemia increases up the age of three years. Efforts to improve strategies for controlling anaemia and malaria are needed.

Objectives Paediatric drug formulations of artemisinin combination therapies and pharmacokinetic data supporting their use in African children are urgently needed for the effective treatment of young children suffering from falciparum malaria in sub-Saharan Africa. Patients and methods In this study, the pharmacokinetic characteristics of a novel paediatric granule formulation of artesunate-mefloquine therapy were evaluated in comparison to the standard tablet formulation in the treatment of uncomplicated malaria in paediatric patients. Twenty-four patients were assigned to treatment according to body weight with either a fixed-dose paediatric granule co-formulation (10-20 kg body weight) or a free-dose co-blister tablet formulation of artesunate-mefloquine (>20-40 kg body weight). Results Median values for C(max) (861 and 930 ng/mL), T(max) (1.5 and 1.5 h) and AUC(0-)(t) (2,050 and 2,470 ng.h/mL) were comparable for dihydroartemisinin in the two groups. Exploratory analysis of mefloquine plasma levels revealed a trend towards higher concentrations in the younger age group during the absorption phase (2,550 and 1,815 ng/mL, 54 h after initiation of treatment, respectively). Median mefloquine concentrations at day 28 were 197 and 343 ng/mL, respectively. Conclusions The pharmacokinetic characteristics of the two paediatric dosage forms, i.e. the novel fixed-dose co-formulation and the standard co-blister of artesunate-mefloquine show comparable results in the two treatment groups. The novel fixed-dose paediatric formulation is an interesting option for outpatient treatment of uncomplicated malaria in African children.

In areas where malaria transmission is stable, infants are often born to mothers who had Plasmodium falciparum infections during pregnancy. A significant number become exposed to infected erythrocytes or soluble parasite products with subsequent fetal immune priming or tolerance in utero. We performed ELISA to asses IgG and IgM seropositivity rates against three PfEMP1 DBL-alpha domains from 42 maternal-cord paired samples obtained at delivery from a hyperendemic area in Gabon. IgG was present in up to 80% of the cord serum samples, while IgM was found in only 20% of the same samples. These levels were not dependent on the parity of the mother or the peripheral and placental infectious status. The presence of IgM against DBL-alpha domain in cord serum samples suggests that this component is able to cross the placental barrier and mount a fetal immune response.

During gestation, inflammatory cytokines are sometimes more abundant than growth-promoting cytokines, and via direct or indirect effects, proinflammatory cytokines lead to intrauterine growth retardation. We used an enzyme-linked immunosorbent assay to measure the concentrations of three proinflammatory cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin-12 (IL-12p40), as well as interleukin-15 (IL-15) and monocyte chemotactic protein-1 (MCP-1), in plasma from peripheral, placental and cord blood of thirty pregnant Gabonese women. All of these women lived in Libreville and Lambaréné, two malaria hyperendemic areas. IL-12p40 concentrations were higher in cord blood than in placental or peripheral blood. The MCP-1 concentration was higher in placental blood, than in peripheral or cord blood. IL-15 concentrations were similar at the three sites. MCP-1 concentrations were higher in the placentas of primiparous women than in those of multiparous women. The highest concentrations were found in infected placentas. IL-15 concentrations were significantly higher in peripheral and placental plasma from uninfected women than in plasma from infected women. Strong positive correlations were found between placental and cord IL-12p40 and IL-15 plasma concentrations. Likewise, a strong positive correlation was found between IL-12p40 and MCP-1 concentrations in cord and peripheral plasma. These results suggest that placental, maternal peripheral and cord blood present different cytokine profiles in response to P. falciparum.

0 d Malaria caused byPlasmodium falciparum is a major pubic health problem with significant economic and social conequences for many developing countries. Estimates of global ncidence of the disease range from 300 to 500 million cases er year, with up to 2.7 million deaths, primarily children in frica [1,2]. Despite years of intensive research, an effective accine is not available and the parasite displays growing esistance to currently available drugs. Rapid progress in geomic research, of the human malaria parasite P. falciparum 3], has stimulated basic research by creating the basis for the dentification and study of parasite proteins that can be used s targets for new drugs and vaccine development. In view f a vaccine for malaria, the construction of a multivalent ubunit vaccine is being considered a major way forward. he selection of protein antigens for such a vaccine has been ampered by the lack of a reliably and readily accessible hallenge system for P. falciparum. Efforts to identify plasodial antigens that trigger protective responses have and ill continue to benefit from studies of rodent malaria parsites [4]. Although, it is argued that these models are quite ifferent from the human system, it is, however, generally agreed that studies in these models can give valuable information in selecting vaccine candidates. The P. yoelii and P. chabaudi models have been effectively used to investigate immunity against blood stage parasites [5,6]. In related studies, two highly enriched membrane proteins of P. yoelii were shown to mediate protective immunity in mice [7,8]. Further studies implicated specific antigens termed pypAg-1 and pypAg-3 characterised by tryptophanrich domains. In a recent study, we identified and characterised a P. falciparum homologue of pypAg-1. Based on its amino acid sequence, we termed it Tryptophan–Threoninerich Antigen (TryThrA), that has typical characteristics of an exported P. falciparum protein [9]. Further searching the malaria database predicted paralogues in P. falciparum. In this study, we identified and characterized the second member of this gene family, which has features similar to the pypAg-3 gene of P. yoelii, which we have termed Merozoiteassociated Tryptophan-rich Antigen (MaTrA). We have also investigated the natural immune responses of TryThrA and MaTrA using serum samples from semi-immune individuals from Lambarene, Gabon.

To investigate if severe malarial anemia is associated with specific cytokine overproduction, we evaluated serum levels of soluble Fas ligand (sFasL), tumor necrosis factor (TNF-alpha) and interleukin-10 (IL-10) from three groups of young children with Plasmodium falciparum infection (asymptomatic cases, uncomplicated malaria cases and severe malarial anemia cases), in a hyperendemic area of Gabon. In uncomplicated cases, only TNF levels were significantly (p0.001) increased in comparison to asymptomatic cases with P. falciparum infection. High levels of sFasL, TNF-alpha and IL-10 were associated with low hemoglobin concentrations, sFasL levels were significantly higher in children with severe malarial anemia (p0.001) as compared to both other groups. The parasite density was positively correlated with IL-10, TNF-alpha and sFasL levels. TNF-alpha and sFasL, but not IL-10 or parasitemia, were independent predictors of hemoglobin concentrations. These results suggest that, in malaria, a specific dysregulation of the cytokine balance may lead to complications such as severe anemia.

In areas where malaria is endemic, pregnancy is associated with increased susceptibility to malaria. It is generally agreed that this risk ends with delivery and decreases with the number of pregnancies. Our study aimed to demonstrate relationships between malarial parasitaemia and age, gravidity and anaemia in pregnant women in Libreville, the capital city of Gabon. Peripheral blood was collected from 311 primigravidae and women in their second pregnancy. Thick blood smears were checked, as were the results of haemoglobin electrophoresis. We also looked for the presence of anaemia, fever, and checked whether the volunteers had had chemoprophylaxis. The study was performed in Gabon where malaria transmission is intense and perennial. A total of 177 women (57%) had microscopic parasitaemia; 139 (64%)of them were primigravidae, 38 (40%) in their second pregnancy and 180 (64%) were teenagers. The parasites densities were also higher in primigravidae and teenagers. The prevalence of anaemia was 71% and was associated with microscopic Plasmodium falciparum parasitaemia: women with moderate or severe anaemia had higher parasite prevalences and densities. However, the sickle cell trait, fever and the use of chemoprophylaxis did not have a significant association with the presence of P. falciparum. These results suggest that the prevalence of malaria and the prevalence of anaemia, whether associated with malaria or not, are higher in pregnant women in Gabon. Primigravidae and young pregnant women are the most susceptible to infection. It is, therefore, urgent to design an effective regimen of malaria prophylaxis for this high risk population.

Background In Gabon, following the adoption of amodiaquine/artesunate combination (AQ/AS) as first-line treatment of malaria and of sulphadoxine/pyrimethamine (SP) for preventive intermittent treatment of pregnant women, a clinical trial of SP versus AQ was conducted in a sub-urban area. This is the first study carried out in Gabon following the WHO guidelines. Methods A random comparison of the efficacy of AQ (10 mg/kg/day × 3 d) and a single dose of SP (25 mg/kg of sulphadoxine/1.25 mg/kg of pyrimethamine) was performed in children under five years of age, with uncomplicated falciparum malaria, using the 28-day WHO therapeutic efficacy test. In addition, molecular genotyping was performed to distinguish recrudescence from reinfection and to determine the frequency of the dhps K540E mutation, as a molecular marker to predict SP-treatment failure. Results The day-28 PCR-adjusted treatment failures for SP and AQ were 11.6% (8/69; 95% IC: 5.5–22.1) and 28.2% (20/71; 95% CI: 17.7–38.7), respectively This indicated that SP was significantly superior to AQ (P = 0.019) in the treatment of uncomplicated childhood malaria and for preventing recurrent infections. Both treatments were safe and well-tolerated, with no serious adverse reactions recorded. The dhps K540E mutation was not found among the 76 parasite isolates tested. Conclusion The level of AQ-resistance observed in the present study may compromise efficacy and duration of use of the AQ/AS combination, the new first-line malaria treatment. Gabonese policy-makers need to plan country-wide and close surveillance of AQ/AS efficacy to determine whether, and for how long, these new recommendations for the treatment of uncomplicated malaria remain valid.