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OBJECTIVES: To determine the impact of insecticide-treated curtains (ITC) on all-cause child mortality (6-59 months) over a period of six years. To determine whether initial reductions in child mortality following the implementation of ITC are sustained over the longer term or whether "delayed" mortality occurs. METHODS: A rural population of ca 100 000 living in an area with high, seasonal Plasmodium falciparum transmission was studied in Burkina Faso. Annual censuses were conducted from 1993 to 2000 to measure child mortality. ITC to cover doors, windows, and eaves were provided to half the population in 1994 with the remainder receiving ITC in 1996. Curtains were re-treated or, if necessary, replaced annually. FINDINGS: Over six years of implementation of ITC, no evidence of the shift in child mortality from younger to older children was observed. Estimates of the reduction in child mortality associated with ITC ranged from 19% to 24%. CONCLUSIONS: In our population there was no evidence to suggest that initial reduction in child mortality associated with the introduction of insecticide-treated materials was subsequently compromised by a shift in child mortality to older-aged children. Estimates of the impact of ITC on child mortality in this population range from 19% to 24%.

Abstract Backgound Treatment of confirmed malaria patients with Artemisinin-based Combination Therapy (ACT) at remote areas is the goal of many anti-malaria programs. Introduction of effective and affordable malaria Rapid Diagnosis Test (RDT) in remote areas could be an alternative tool for malaria case management. This study aimed to assess performance of the OptiMAL dipstick for rapid malaria diagnosis in children under five. Methods Malaria symptomatic and asymptomatic children were recruited in a passive manner in two community clinics (CCs). Malaria diagnosis by microscopy and RDT were performed. Performance of the tests was determined. Results RDT showed similar ability (61.2%) to accurately diagnose malaria as microscopy (61.1%). OptiMAL showed a high level of sensitivity and specificity, compared with microscopy, during both transmission seasons (high & low), with a sensitivity of 92.9% vs. 74.9% and a specificity of 77.2% vs. 87.5%. Conclusion By improving the performance of the test through accurate and continuous quality control of the device in the field, OptiMAL could be suitable for use at CCs for the management and control of malaria.

Abstract Background Genetic factors play a key role in determining resistance/susceptibility to infectious disease. Susceptibility of the human host to malaria infection has been reported to be influenced by genetic factors, which could be confounders if not taken into account in the assessment of the efficacy of interventions against malaria. This study aimed to assess the relationship between haemoglobin genotypes and malaria in children under five years in a site being characterized for future malaria vaccine trials. Methods The study population consisted of 452 children living in four rural villages. Hb genotype was determined at enrolment. Clinical malaria incidence was evaluated over a one-year period using combined active and passive surveillance. Prevalence of infection was evaluated via bi-annual cross-sectional surveys. At each follow-up visit, children received a brief clinical examination and thick and thin blood films were prepared for malaria diagnosis. A clinical malaria was defined as Plasmodium falciparum parasitaemia >2,500 parasites/μl and axillary temperature ≥37.5°C or reported fever over the previous 24 hours. Results Frequencies of Hb genotypes were 73.2% AA; 15.0% AC; 8.2% AS; 2.2% CC; 1.1% CS and 0.2% SS. Prevalence of infection at enrolment ranged from 61.9%-54.1% among AA, AC and AS children. After one year follow-up, clinical malaria incidence (95% CI) (episodes per person-year) was 1.9 (1.7-2.0) in AA, 1.6 (1.4-2.1) in AC, and 1.7 (1.4-2.0) in AS children. AC genotype was associated with lower incidence of clinical malaria relative to AA genotype among children aged 1–2 years [rate ratio (95% CI) 0.66 (0.42-1.05)] and 2–3 years [rate ratio (95% CI) 0.37 (0.18-0.75)]; an association of opposite direction was however apparent among children aged 3–4 years. AS genotype was associated with lower incidence of clinical malaria relative to AA genotype among children aged 2–3 years [rate ratio (95% CI) 0.63 (0.40-1.01)]. Conclusions In this cohort of children, AC or AS genotype was associated with lower risk of clinical malaria relative to AA genotype only among children aged one to three years. It would be advisable for clinical studies of malaria in endemic regions to consider haemoglobin gene differences as a potentially important confounder, particularly among younger children.

Abstract Background Patient immune status is thought to affect the efficacy of anti-malarial chemotherapy. This is a subject of some importance, since evidence of immunity-related interactions may influence our use of chemotherapy in populations with drug resistance, as well as assessment of the value of suboptimal vaccines. The study aim was to investigate relationship between antibodies and anti-malarial drug treatment outcomes. Methods Some 248 children aged 0.5 and 15 years were recruited prior to the high malaria transmission season. Venous blood (5 ml) was obtained from each child to measure antibody levels to selected malaria antigens, using ELISA. Blood smears were also performed to assess drug efficacy and malaria infection prevalence. Children were actively followed up to record clinical malaria cases. Results IgG levels to MSP3 were always higher in the successfully treated group than in the group with treatment failure. The same observation was made for GLURP but the reverse observation was noticed for MSP1-19. Cytophilic and non-cytophilic antibodies were significantly associated with protection against all three antigens, except for IgG4 to MSP1-19 and GLURP. Conclusion Acquired anti-malarial antibodies may play an important role in the efficacy of anti-malarial drugs in younger children more susceptible to the disease.

Abstract Background Fulani ethnic group individuals are less susceptible than sympatric Mossi ethnic group, in term of malaria infection severity, and differ in antibody production against malaria antigens. The differences in susceptibility to malaria between Fulani and Mossi ethnic groups are thought to be regulated by different genetic backgrounds and offer the opportunity to compare haematological parameters, Tregs and γδT cell profiles in seasonal and stable malaria transmission settings in Burkina Faso. The study was conducted at two different time points i.e. during the high and low malaria transmission period. Results Two cross-sectional surveys were undertaken in adults above 20 years belonging either to the Fulani or the Mossi ethnic groups 1) at the peak of the malaria transmission season and 2) during the middle of the low malaria transmission season. Full blood counts, proportions of Tregs and γδ T cells were measured at both time-points. As previously shown the Fulani and Mossi ethnic groups showed a consistent difference in P. falciparum infection rates and parasite load. Differential white blood cell counts showed that the absolute lymphocyte counts were higher in the Mossi than in the Fulani ethnic group at both time points. While the proportion of CD4+CD25high was higher in the Fulani ethnic group at the peak of malaria transmission season (p = 0.03), no clear pattern emerged for T regulatory cells expressing FoxP3+ and CD127low. However CD3+γδ+ subpopulations were found to be higher in the Fulani compared to the Mossi ethnic group, and this difference was statistically significant at both time-points (p = 0.004 at low transmission season and p = 0.04 at peak of transmission). Conclusion Our findings on regulatory T cell phenotypes suggest an interesting role for immune regulatory mechanisms in response to malaria. The study also suggests that TCRγδ + cells might contribute to the protection against malaria in the Fulani ethnic group involving their reported parasite inhibitory activities.

Abstract Background The Anopheles gambiae gSG6 is an anopheline-specific salivary protein which helps female mosquitoes to efficiently feed on blood. Besides its role in haematophagy, gSG6 is immunogenic and elicits in exposed individuals an IgG response, which may be used as indicator of exposure to the main African malaria vector A. gambiae. However, malaria transmission in tropical Africa is sustained by three main vectors (A. gambiae, Anopheles arabiensis and Anopheles funestus) and a general marker, reflecting exposure to at least these three species, would be especially valuable. The SG6 protein is highly conserved within the A. gambiae species complex whereas the A. funestus homologue, fSG6, is more divergent (80% identity with gSG6). The aim of this study was to evaluate cross-reactivity of human sera to gSG6 and fSG6. Methods The A. funestus SG6 protein was expressed/purified and the humoral response to gSG6, fSG6 and a combination of the two antigens was compared in a population from a malaria hyperendemic area of Burkina Faso where both vectors were present, although with a large A. gambiae prevalence (>75%). Sera collected at the beginning and at the end of the high transmission/rainy season, as well as during the following low transmission/dry season, were analysed. Results According to previous observations, both anti-SG6 IgG level and prevalence decreased during the low transmission/dry season and showed a typical age-dependent pattern. No significant difference in the response to the two antigens was found, although their combined use yielded in most cases higher IgG level. Conclusions Comparative analysis of gSG6 and fSG6 immunogenicity to humans suggests the occurrence of a wide cross-reactivity, even though the two proteins carry species-specific epitopes. This study supports the use of gSG6 as reliable indicator of exposure to the three main African malaria vectors, a marker which may be useful to monitor malaria transmission and evaluate vector control measures, especially in conditions of low malaria transmission and/or reduced vector density. The Anopheles stephensi SG6 protein also shares 80% identity with gSG6, suggesting the attractive possibility that the A. gambiae protein may also be useful to assess human exposure to several Asian malaria vectors.

Abstract Background The weekly chemoprophylaxis of malaria during pregnancy with chloroquine (CQ) has become problematic with the increasing resistance of Plasmodium falciparum to this drug. There was a need to test the benefits of new strategies over the classical chemoprophylaxis. This study was conducted to provide data to the National Malarial Control Programme for an evidence-based policy change decision making process. It compares the efficacy of two IPT regimens, using chloroquine (CQ) or sulphadoxine/pyrimethamine (SP), with the classical chemoprophylaxis regimen using CQ in reducing the adverse outcomes of malaria infection, for the mother and the foetus. Methods Pregnant women attending the first antenatal care visit were randomly assigned to one of the three treatment regimens. They were subsequently followed up till delivery. Maternal, placental and cord blood samples were obtained upon delivery to check for P. falciparum infection. Results A total of 648 pregnant women were enrolled in the study. Delivery outcome were available for 423 of them. Peripheral maternal P. falciparum infection at delivery was found in 25.8% of the women. The proportion of women with maternal infection was significantly lower in the IPTp/SP group than in the CQ group (P << 0.000). The prevalence of placental malaria was 18.8% in the CWC/CQ group; 15.9% in the IPTp/CQ group and 10.6% in the IPTp/SP group. The incidence of LBW (weigth < 2,500 g) was significantly higher among infants of mothers in the CWC/CQ group (23.9%) as compared with those of mothers in the IPTp/CQ (15.6%) and IPTp/SP (11.6%) groups (p = 0.02) Conclusion Intermittent preventive treatment with SP has shown clear superiority in reducing adverse outcomes at delivery, as compared with intermittent preventive treatment with CQ and classical chemoprophylaxis with CQ.

Abstract Background The clinical presentation of malaria, considered as the result of a complex interaction between parasite and human genetics, is described to be different between rural and urban areas. The analysis of the Plasmodium falciparum genetic diversity in children with uncomplicated malaria, living in these two different areas, may help to understand the effect of urbanization on the distribution of P. falciparum genotypes. Methods Isolates collected from 75 and 89 children with uncomplicated malaria infection living in a rural and an urban area of Burkina Faso, respectively, were analysed by a nested PCR amplification of msp1 and msp2 genes to compare P. falciparum diversity. Results The K1 allelic family was widespread in children living in the two sites, compared to other msp1 allelic families (frequency >90%). The MAD 20 allelic family of msp1 was more prevalent (p = 0.0001) in the urban (85.3%) than the rural area (63.2%). In the urban area, the 3D7 alleles of msp2 were more prevalent compared to FC27 alleles, with a high frequency for the 3D7 300bp allele (>30%). The multiplicity of infection was in the range of one to six in the urban area and of one to seven in the rural area. There was no difference in the frequency of multiple infections (p = 0.6): 96.0% (95% C.I: 91.6–100) in urban versus 93.1% (95%C.I: 87.6–98.6) in rural areas. The complexity of infection increased with age [p = 0.04 (rural area), p = 0.06 (urban area)]. Conclusion Urban-rural area differences were observed in some allelic families (MAD20, FC27, 3D7), suggesting a probable impact of urbanization on genetic variability of P. falciparum. This should be taken into account in the implementation of malaria control measures.

The estimates of enterotoxigenic Escherichia coli (ETEC) and Shigella burden in developing countries are limited by the lack of rapid, accessible, and sensitive diagnostics and surveillance tools. We used a "Rapid LAMP based Diagnostic Test (RLDT)" to detect ETEC and Shigella in diarrheal and non-diarrheal stool samples from a 12-month longitudinal cohort of children under five years of age in a peri-urban area of Ouagadougou in Burkina Faso (West Africa). To allow comparison with the RLDT- Shigella results, conventional culture methods were used to identify Shigella strains in the stool samples. As conventional culture alone cannot detect ETEC cases, a subset of E. coli -like colonies was tested using conventional PCR to detect ETEC toxins genes. Of the 165 stool samples analyzed for ETEC, 24.9% were positive when using RLDT against 4.2% when using culture followed by PCR. ETEC toxin distribution when using RLDT was STp 17.6% (29/165), LT 11.5% (19/165), and STh 8.5% (14/165). Of the 263 specimens tested for Shigella , 44.8% were positive when using RLDT against 23.2% when using culture. The sensitivity and specificity of the RLDT compared to culture (followed by PCR for ETEC) were 93.44% and 69.8% for Shigella and 83.7% and 77.9% for ETEC, respectively. This study indicates that both Shigella and ETEC are substantially underdiagnosed when using conventional culture and highlights the potential contribution of the new RLDT method to improve enteric disease burden estimation and to guide future efforts to prevent and control bacterial enteric infection and disease.

We assessed anti-Vi IgG/IgA responses to typhoid conjugate vaccine (TCV) in children enrolled in a double-blind randomized controlled, phase 2 trial in Burkina Faso. Anti-Vi IgG seroconversion and anti-Vi IgA titers were higher in TCV than control recipients at 30-35 months post-vaccination. TCV induces durable immunity in Burkinabe children vaccinated at 15 months., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society.)

Continuous monitoring of malaria epidemiology is needed in malaria-endemic settings to inform malaria control and elimination strategies. This study aimed to compare the malariometric indices between the under-fives and school-age children. We surveyed children aged 1.5 to 12 years for plasmodia carriage with the aim of including them in a longitudinal follow-up cohort. The survey took place from 7-11 September 2020 in a southwest area of Burkina Faso. Clinical and demographic data including malaria control measures were collected. A finger prick blood sample was taken for haemoglobin testing, and blood smears and dried blood spot preparation. The malariometric indices were calculated and compared between school-age children and those under the age of five. Multiple logistic regression was fitted to assess the association between malaria parasite carriage and age categories. Based on the PCR results, the parasite prevalence was 21.4% in the under-fives versus 44.2% in school-age children ( p -value < 0.0001), with a pooled prevalence of 32.7% (CI = [28.8, 36.8]). The gametocyte prevalence was also significantly higher in school-age children (11.9%) compared to the under-fives (3.7%). Adjusted for covariates, school-age children were 2.9 times (IC = [2.0, 4.2]) more likely to carry the asexual parasite, compared to the under-fives. Malaria was moderate and stable endemic in this area and school-age children play a key role in the spread of the disease. The WHO conditional recommendation for intermittent preventive treatment of malaria in school-aged children living in malaria-endemic settings with moderate to high perennial or seasonal transmission should be implemented.

Antimalarial drugs that can block the transmission of Plasmodium gametocytes to mosquito vectors would be highly beneficial for malaria elimination efforts. Identifying transmission-blocking drugs currently relies on evaluation of their activity against gametocyte-producing laboratory parasite strains and would benefit from a testing pipeline with genetically diverse field isolates. The aims of this study were to develop a pipeline to test drugs against P. falciparum gametocyte field isolates and to evaluate the transmission-blocking activity of a set of novel compounds. Two assays were designed so they could identify both the overall transmission-blocking activity of a number of marketed and experimental drugs by direct membrane feeding assays (DMFA), and then also discriminate between those that are active against the gametocytes (gametocyte killing or sterilizing) or those that block development in the mosquito (sporontocidal). These DMFA assays used venous blood samples from naturally infected Plasmodium falciparum gametocyte carriers and locally reared Anopheles gambiae s.s. mosquitoes. Overall transmission-blocking activity was assessed following a 24 hour incubation of compound with gametocyte infected blood (TB-DMFA). Sporontocidal activity was evaluated following addition of compound directly prior to feeding, without incubation (SPORO-DMFA); Gametocyte viability was retained during 24-hour incubation at 37°C when gametocyte infected red blood cells were reconstituted in RPMI/serum. Methylene-blue, MMV693183, DDD107498, atovaquone and P218 showed potent transmission-blocking activity in the TB-DMFA, and both atovaquone and the novel antifolate P218 were potent inhibitors of sporogonic development in the SPORO-DMA. This work establishes a pipeline for the integral use of field isolates to assess the transmission-blocking capacity of antimalarial drugs to block transmission that should be validated in future studies., Competing Interests: No, the authors have declared that no competing interests exist., (Copyright: © 2023 B. Henry et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The experimental malaria vaccine ChAd63 MVA ME-TRAP previously showed protective efficacy against Plasmodium falciparum infection in Phase IIa sporozoite challenge studies in adults in the United Kingdom and in a Phase IIb field efficacy trial in Kenyan adults. However, it failed to demonstrate efficacy in a phase IIb trial in 5-17 month-old children in an area of high malaria transmission in Burkina Faso. This secondary analysis investigated whether exposure to malaria or nutritional status might be associated with reduced responses to vaccination in this cohort. Parasite blood smears and anti-AMA-1 IgG titres were used to assess history of exposure to malaria and weight-for-length Z scores were calculated to assess nutritional status. Differences in vaccine-specific anti-TRAP IgG titre and ex vivo IFNγ ELISpot response were measured between groups. In total, n = 336 volunteers randomised to receive the experimental vaccine regimen were included in this analysis. A positive smear microscopy result was associated with reduced anti-TRAP IgG titre (geometric mean titre: 2775 (uninfected) vs 1968 (infected), p = 0.025), whilst anti-AMA-1 IgG titres were weakly negatively correlated with reduced ex vivo IFNγ ELISpot response (r = -0.18, p = 0.008). Nutritional status was not associated with either humoral or cellular immunogenicity. Vaccine efficacy was also measured separately for vaccinees with positive and negative blood smears. Although not significant in either group compared to controls, vaccine efficacy measured by Cox hazard ratio was higher in uninfected compared to infected individuals (19.8% [ p = 0.50] vs 3.3% [ p = 0.69]). Overall, this data suggests exposure to malaria may be associated with impaired vaccine immunogenicity. This may have consequences for the testing and eventual deployment of various vaccines, in areas with high endemicity for malaria., Trial Registration: Pactr.org, identifier PACTR201208000404131; ClinicalTrials.gov, identifier NCT01635647., Competing Interests: AH is a named inventor on patent applications and issued patents relating to malaria vectored vaccines and immunization regimes. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Morter, Tiono, Nébié, Hague, Ouedraogo, Diarra, Viebig, Hill, Ewer and Sirima.)

Background: Malaria in pregnancy remains a public health problem in sub-Saharan Africa. Identifying risk factors for malaria in pregnancy could assist in developing interventions to reduce the risk of malaria in Burkina Faso and other countries in the region., Methods: Two cross-sectional surveys were carried out to measure Plasmodium falciparum infection using microscopy in pregnant women in Saponé Health District, central Burkina Faso. Data were collected on individual, household and environmental variables and their association with P. falciparum infection assessed using multivariable analysis., Results: A total of 356 pregnant women were enrolled in the surveys, 174 during the dry season and 182 during the wet season. The mean number of doses of sulfadoxine-pyrimethamine for Intermittent Preventive Treatment in pregnancy (IPTp-SP) was 0.4 doses during the first trimester, 1.1 doses at the second and 2.3 doses at the third. Overall prevalence of P. falciparum infection by microscopy was 15.7%; 17.8% in the dry season and 13.7% in the wet season. 88.2% of pregnant women reported sleeping under an insecticide-treated net (ITN) on the previous night. The odds of P. falciparum infection was 65% lower in women who reported using an ITN compared to those that did not use an ITN (Odds ratio, OR = 0.35, 95% CI 0.14-0.86, p = 0.02). IPTp-SP was also associated with reduced P. falciparum infection, with each additional dose of IPTp-SP reducing the odds of infection by 44% (OR = 0.56, 95% CI 0.39-0.79, p = 0.001). Literate women had a 2.54 times higher odds of P. falciparum infection compared to illiterate women (95% CI 1.31-4.91, p = 0.006)., Conclusions: The prevalence of P. falciparum infection among pregnant women remains high in Burkina Faso, although use of IPTp-SP and ITNs were found to reduce the odds of infection. Despite this, compliance with IPTp-SP remains far from that recommended by the National Malaria Control Programme and World Health Organization. Behaviour change communication should be strengthened to encourage compliance with protective malaria control tools during pregnancy., (© 2021. The Author(s).)

Objectives: In 2017, the World Health Organisation (WHO) pre-qualified a single-dose typhoid conjugate vaccine (TCV) and identified TCV co-administration studies as a research priority. Accordingly, we tested co-administration of Typbar TCV® (Bharat Biotech International) with measles-rubella (MR) and yellow fever (YF) vaccines., Methods: We conducted a randomized, double-blind, and controlled, phase 2 trial in Ouagadougou, Burkina Faso. Healthy children aged 9-11 months were randomized 1:1 to receive TCV (Group 1) or control vaccine (inactivated polio vaccine (IPV), Group 2). Vaccines were administered intramuscularly with routine MR and YF vaccines. Safety was assessed by (1) local and systemic reactions on days 0, 3, and 7; (2) unsolicited adverse events within 28 days; and (3) serious adverse events (SAEs) within six months after immunization., Results: We enrolled, randomized, and vaccinated 100 eligible children (49 Group 1 and 51 Group 2). Safety outcomes occurred with similar frequency in both groups: local/solicited reactions (Group 1: 1/49, Group 2: 3/50), systemic/solicited reactions (Group 1: 4/49, Group 2: 9/50), unsolicited adverse events (Group 1: 26/49, Group 2: 33/51), and SAEs (Group 1: 2/49, Group 2: 3/51). TCV conferred robust immunogenicity without interference with MR or YF vaccines., Conclusion: TCV can be safely co-administered with MR and YF vaccines to children at the 9-month vaccination visit., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Plasmodium falciparum gametocyte kinetics and infectivity may differ between chronic and incident infections. In the current study, we assess parasite kinetics and infectivity to mosquitoes among children (aged 5-10 years) from Burkina Faso with (a) incident infections following parasite clearance (n = 48) and (b) chronic asymptomatic infections (n = 60). In the incident infection cohort, 92% (44/48) of children develop symptoms within 35 days, compared to 23% (14/60) in the chronic cohort. All individuals with chronic infection carried gametocytes or developed them during follow-up, whereas only 35% (17/48) in the incident cohort produce gametocytes before becoming symptomatic and receiving treatment. Parasite multiplication rate (PMR) and the relative abundance of ap2-g and gexp-5 transcripts are positively associated with gametocyte production. Antibody responses are higher and PMR lower in chronic infections. The presence of symptoms and sexual stage immune responses are associated with reductions in gametocyte infectivity to mosquitoes. We observe that most incident infections require treatment before the density of mature gametocytes is sufficient to infect mosquitoes. In contrast, chronic, asymptomatic infections represent a significant source of mosquito infections. Our observations support the notion that malaria transmission reduction may be expedited by enhanced case management, involving both symptom-screening and infection detection.

Primaquine (PQ) is an antimalarial drug with the potential to reduce malaria transmission due to its capacity to clear mature Plasmodium falciparum gametocytes in the human host. However, the large-scale roll-out of PQ has to be counterbalanced by the additional risk of drug-induced hemolysis in individuals suffering from Glucose-6-phospate dehydrogenase (G6PD) deficiency, a genetic condition determined by polymorphisms on the X-linked G6PD gene. Most studies on G6PD deficiency and PQ-associated hemolysis focused on the G6PD A- variant, a combination of the two single nucleotide changes G202A (rs1050828) and A376G (rs1050829), although other polymorphisms may play a role. In this study, we tested the association of 20 G6PD single nucleotide polymorphisms (SNPs) with hemolysis measured seven days after low single dose of PQ given at the dose of 0.1 mg/kg to 0.75 mg/kg in 957 individuals from 6 previously published clinical trials investigating the safety and efficacy of this drug spanning five African countries. After adjusting for inter-study effects, age, gender, baseline hemoglobin level, PQ dose, and parasitemia at screening, our analysis showed putative association signals from the common G6PD mutation, A376G [-log 10 ( p -value) = 2.44] and two less-known SNPs, rs2230037 [-log 10 ( p -value] = 2.60), and rs28470352 [-log 10 ( p -value) = 2.15]; A376G and rs2230037 were in very strong linkage disequilibrium with each other ( R 2 = 0.978). However, when the effects of these SNPs were included in the same regression model, the subsequent associations were in the borderline of statistical significance. In conclusion, whilst a role for the A- variant is well established, we did not observe an important additional role for other G6PD polymorphisms in determining post-treatment hemolysis in individuals treated with low single-dose PQ., Competing Interests: JC and LM were employed by the company LGC Genomics. The remaining authors declare that the research was conducted in the absence of any commercial and financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sepúlveda, Grignard, Curry, Mahey, Bastiaens, Tiono, Okebe, Coulibaly, Gonçalves, Affara, Ouédraogo, Bougouma, Sanou, Nébié, Lanke, Sirima, Dicko, d’Alessandro, Clark, Campino, Chen, Eziefula, Gosling, Bousema and Drakeley.)

Objectives: The World Health Organization pre-qualified single-dose typhoid conjugate vaccine (TCV) and requested data on co-administration with routine vaccines. The co-administration of Typbar TCV (Bharat Biotech International) with routine group A meningococcal conjugate vaccine (MCV-A) and measles-rubella (MR) vaccine was tested., Methods: This was a double-blind, randomized controlled trial performed in Ouagadougou, Burkina Faso. Children were recruited at the 15-month vaccination visit and were assigned randomly (1:1:1) to three groups. Group 1 children received TCV plus control vaccine (inactivated polio vaccine) and MCV-A 28 days later; group 2 children received TCV and MCV-A; group 3 children received MCV-A and control vaccine. Routine MR vaccine was administered to all participants. Safety was assessed at 0, 3, and 7 days after immunization, and unsolicited adverse events and serious adverse events were assessed for 28 days and 6 months after immunization, respectively., Results: A total of 150 children were recruited and vaccinated. Solicited symptoms were infrequent and similar for TCV and control recipients, as were adverse events (group 1, 61.2%; group 2, 64.0%; group 3, 68.6%) and serious adverse events (group 1, 2.0%; group 2, 8.0%; group 3, 5.9%). TCV generated robust immunity without interference with MCV-A vaccine., Conclusions: TCV can be safely co-administered at 15 months with MCV-A without interference. This novel study on the co-administration of TCV with MCV-A provides data to support large-scale uptake in sub-Saharan Africa., (Published by Elsevier Ltd.)

Background: PRIMVAC is a VAR2CSA-derived placental malaria vaccine candidate aiming to prevent serious clinical outcomes of Plasmodium falciparum infection during pregnancy. We assessed the safety and immunogenicity of PRIMVAC adjuvanted with Alhydrogel or glucopyranosyl lipid adjuvant in stable emulsion (GLA-SE) in French and Burkinabe women who were not pregnant., Methods: This first-in-human, randomised, double-blind, placebo-controlled, dose escalation trial was done in two staggered phases, a phase 1A trial in 18-35-year-old women who were malaria naive in a hospital in France and a subsequent phase 1B trial in women who were naturally exposed to P falciparum and nulligravid in the clinical site of a research centre in Burkina Faso. Volunteers were recruited into four sequential cohorts receiving PRIMVAC intramuscularly at day 0, 28, and 56: two cohorts in France receiving 20 μg or 50 μg of PRIMVAC and then two in Burkina Faso receiving 50 μg or 100 μg of PRIMVAC. Volunteers were randomly assigned (1:1) to two groups (PRIMVAC adjuvanted with either Alhydrogel or GLA-SE) in France and randomly assigned (2:2:1) to three groups (PRIMVAC adjuvanted with either Alhydrogel, GLA-SE, or placebo) in Burkina Faso. Randomisation was centralised, using stratification by cohort and blocks of variable size, and syringes were masked by opaque labels. The primary endpoint was the proportion of participants with any grade 3 or higher adverse reaction to vaccination up until day 35. Safety at later time points as well as humoral and cellular immunogenicity were assessed in secondary endpoints. This trial is registered with ClinicalTrials.gov, NCT02658253., Findings: Between April 19, 2016, and July 13, 2017, 68 women (18 in France, 50 in Burkina Faso) of 101 assessed for eligibility were included. No serious adverse event related to the vaccine occurred. PRIMVAC antibody titres increased with each dose and seroconversion was observed in all women vaccinated with PRIMVAC (n=57). PRIMVAC antibody titres reached a peak (geometric mean 11 843·0, optical density [OD] 1·0, 95% CI 7559·8-18 552·9 with 100 μg dose and GLA-SE) 1 week after the third vaccination (day 63). Compared with Alhydrogel, GLA-SE tended to improve the PRIMVAC antibody response (geometric mean 2163·5, OD 1·0, 95% CI 1315·7-3557·7 with 100 μg dose and Alhydrogel at day 63). 1 year after the last vaccination, 20 (71%) of 28 women who were vaccinated with PRIMVAC/Alhydrogel and 26 (93%) of 28 women who were vaccinated with PRIMVAC/GLA-SE still had anti-PRIMVAC antibodies, although antibody magnitude was markedly lower (452·4, OD 1·0, 95% CI 321·8-636·1 with 100 μg dose and GLA-SE). These antibodies reacted with native homologous VAR2CSA expressed by NF54-CSA infected erythrocytes (fold change from baseline at day 63 with 100 μg dose and GLA-SE: 10·74, 95% CI 8·36-13·79). Limited cross-recognition, restricted to sera collected from women that received the 100 μg PRIMVAC dose, was observed against heterologous VAR2CSA variants expressed by FCR3-CSA (fold change from baseline at day 63: 1·49, 95% CI 1·19-1·88) and 7G8-CSA infected erythrocytes (1·2, 1·08-1·34)., Interpretation: PRIMVAC adjuvanted with Alhydrogel or GLA-SE had an acceptable safety profile, was immunogenic, and induced functional antibodies reacting with the homologous VAR2CSA variant expressed by NF54-CSA infected erythrocytes. Cross-reactivity against heterologous VAR2CSA variants was limited and only observed in the higher dose group. An alternate schedule of immunisation, antigen dose, and combinations with other VAR2CSA-based vaccines are envisaged to improve the cross-reactivity against heterologous VAR2CSA variants., Funding: Bundesministerium für Bildung und Forschung, through Kreditanstalt für Wiederaufbau, Germany; Inserm, and Institut National de Transfusion Sanguine, France; Irish Aid, Department of Foreign Affairs and Trade, Ireland., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Individuals living in malaria-endemic regions develop immunity against severe malaria, but it is unclear whether immunity against pre-erythrocytic stages that blocks initiation of blood-stage infection after parasite inoculation develops following continuous natural exposure. Methods: We cleared schoolchildren living in an area (health district of Saponé, Burkina Faso) with highly endemic seasonal malaria of possible sub-patent infections and examined them weekly for incident infections by nested PCR. Plasma samples collected at enrolment were used to quantify antibodies to the pre-eryhrocytic-stage antigens circumsporozoite protein (CSP) and Liver stage antigen 1 (LSA-1). In vitro sporozoite gliding inhibition and hepatocyte invasion inhibition by naturally acquired antibodies were assessed using Plasmodium falciparum NF54 sporozoites. Associations between antibody responses, functional pre-erythrocytic immunity phenotypes and time to infection detected by 18S quantitative PCR were studied. Results: A total of 51 children were monitored. Anti-CSP antibody titres showed a positive association with sporozoite gliding motility inhibition (P<0.0001, Spearman's ρ=0.76). In vitro hepatocyte invasion was inhibited by naturally acquired antibodies (median inhibition, 19.4% [IQR 15.2-40.9%]), and there were positive correlations between invasion inhibition and gliding inhibition (P=0.005, Spearman's ρ=0.67) and between invasion inhibition and CSP-specific antibodies (P=0.002, Spearman's ρ=0.76). Survival analysis indicated longer time to infection in individuals displaying higher-than-median sporozoite gliding inhibition activity (P=0.01), although this association became non-significant after adjustment for blood-stage immunity (P = 0.06). Conclusions: In summary, functional antibodies against the pre-erythrocytic stages of malaria infection are acquired in children who are repeatedly exposed to Plasmodium parasites. This immune response does not prevent them from becoming infected during a malaria transmission season, but might delay the appearance of blood stage parasitaemia. Our approach could not fully separate the effects of pre-erythrocytic-specific and blood-stage-specific antibody-mediated immune responses in vivo ; epidemiological studies powered and designed to address this important question should become a research priority., Competing Interests: No competing interests were disclosed.

Background: Glucose-6-phosphate dehydrogenase deficiency (G6PDd), haemoglobin C (HbC) and S (HbS) are inherited blood disorders (IBD) common in populations in malaria endemic areas. All are associated to some degree with protection against clinical malaria whilst additionally G6PDd is associated with haemolysis following treatment with 8-aminoquinolines. Measuring the prevalence of these inherited blood disorders in affected populations can improve understanding of disease epidemiology. Current methodologies in epidemiological studies commonly rely on individual target amplification and visualization; here a method is presented to simultaneously detect the polymorphisms and that can be expanded to include other single nucleotide polymorphisms (SNPs) of interest., Methods: Human DNA from whole blood samples was amplified in a novel, multiplex PCR reaction and extended with SNP-specific probes in an allele specific primer extension (ASPE) to simultaneously detect four epidemiologically important human markers including G6PD SNPs (G202A and A376G) and common haemoglobin mutations (HbS and HbC). The products were hybridized to magnetic beads and the median fluorescence intensity (MFI) was read on MAGPIX ® (Luminex corp.). Genotyping data was compared to phenotypical data generated by flow cytometry and to established genotyping methods., Results: Seventy-five samples from Burkina Faso (n = 75/78, 96.2%) and 58 samples from The Gambia (n = 58/61, 95.1%) had a G6PD and a HBB genotype successfully assigned by the bead-based assay. Flow cytometry data available for n = 61 samples further supported the concordance between % G6PD normal/deficient cells and genotype., Conclusions: The bead based assay compares well to alternative measures of genotyping and phenotyping for G6PD. The screening is high throughput, adaptable to inclusion of multiple targets of interest and easily standardized.

Background: Heterologous prime boost immunization with chimpanzee adenovirus 63 (ChAd63) and Modified Vaccinia Virus Ankara (MVA) vectored vaccines is a strategy previously shown to provide substantial protective efficacy against P. falciparum infection in United Kingdom adult Phase IIa sporozoite challenge studies (approximately 20-25% sterile protection with similar numbers showing clear delay in time to patency), and greater point efficacy in a trial in Kenyan adults., Methodology: We conducted the first Phase IIb clinical trial assessing the safety, immunogenicity and efficacy of ChAd63 MVA ME-TRAP in 700 healthy malaria exposed children aged 5-17 months in a highly endemic malaria transmission area of Burkina Faso., Results: ChAd63 MVA ME-TRAP was shown to be safe and immunogenic but induced only moderate T cell responses (median 326 SFU/106 PBMC (95% CI 290-387)) many fold lower than in previous trials. No significant efficacy was observed against clinical malaria during the follow up period, with efficacy against the primary endpoint estimate by proportional analysis being 13.8% (95%CI -42.4 to 47.9) at sixth month post MVA ME-TRAP and 3.1% (95%CI -15.0 to 18.3; p = 0.72) by Cox regression., Conclusions: This study has confirmed ChAd63 MVA ME-TRAP is a safe and immunogenic vaccine regimen in children and infants with prior exposure to malaria. But no significant protective efficacy was observed in this very highly malaria-endemic setting., Trial Registration: ClinicalTrials.gov NCT01635647. Pactr.org PACTR201208000404131., Competing Interests: AVSH is a named inventor on patent applications and issued patents relating to malaria vectored vaccines and immunization regimes. This does not alter the author’s adherence to all the PLOS ONE policies on sharing data and materials.

Background: Individuals living in malaria-endemic regions develop naturally acquired immunity against severe malarial disease, but it is unclear whether immunity that affects the establishment of infections develops following continuous natural exposure. Methods: We cleared schoolchildren in Burkina Faso of possible sub-patent infections and examined them weekly for incident infections by PCR. Plasma samples collected at enrolment were used to quantify antibodies to the pre-eryhrocytic-stage antigens circumsporozoite protein (CSP) and liver stage antigen. Sporozoite gliding inhibition by naturally acquired antibodies was assessed using Plasmodium falciparum NF54 sporozoites; hepatocyte invasion was assessed using the human HC-04 hepatoma cell line and NF54 sporozoites. The associations between these functional pre-erythrocytic immunity phenotypes and time to PCR-detected infection were studied. Results: A total of 51 children were monitored; the median time to first detection of infection by PCR or development of clinical symptoms was 28 days. Anti-CSP antibody titres showed a strong positive association with sporozoite gliding motility inhibition (P<0.0001, Spearman's ρ=0.76). In vitro hepatocyte invasion was inhibited by naturally acquired antibodies (median invasion inhibition, 19.4% [IQR 15.2-40.9%]), and there was a positive correlation between gliding and invasion inhibition (P=0.02, Spearman's ρ=0.60). Survival analysis indicated longer time to infection in individuals displaying higher-than-median sporozoite gliding inhibition activity (P=0.01). Conclusions: In summary, functional antibodies against the pre-erythrocytic stages of malaria infection are acquired in children who are repeatedly exposed to Plasmodium parasites. This immune response does not prevent them from becoming infected during a malaria transmission season, but might delay the appearance of blood stage parasitaemia and consequently needs to be considered in the evaluation of malaria vaccines., Competing Interests: No competing interests were disclosed.

Background: Primaquine (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 Findings: In 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., Conclusions: Single 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 Registration: Clinicaltrials.gov NCT02174900 Clinicaltrials.gov NCT02654730.

A detailed understanding of the human infectious reservoir is essential for improving malaria transmission-reducing interventions. Here we report a multi-regional assessment of population-wide malaria transmission potential based on 1209 mosquito feeding assays in endemic areas of Burkina Faso and Kenya. Across both sites, we identified 39 infectious individuals. In high endemicity settings, infectious individuals were identifiable by research-grade microscopy (92.6%; 25/27), whilst one of three infectious individuals in the lowest endemicity setting was detected by molecular techniques alone. The percentages of infected mosquitoes in the different surveys ranged from 0.05 (4/7716) to 1.6% (121/7749), and correlate positively with transmission intensity. We also estimated exposure to malaria vectors through genetic matching of blood from 1094 wild-caught bloodfed mosquitoes with that of humans resident in the same houses. Although adults transmitted fewer parasites to mosquitoes than children, they received more mosquito bites, thus balancing their contribution to the infectious reservoir.

Heterologous prime-boosting with viral vectors encoding the pre-erythrocytic antigen thrombospondin-related adhesion protein fused to a multiple epitope string (ME-TRAP) induces CD8 + T cell-mediated immunity to malaria sporozoite challenge in European malaria-naive and Kenyan semi-immune adults. This approach has yet to be evaluated in children and infants. We assessed this vaccine strategy among 138 Gambian and Burkinabe children in four cohorts: 2- to 6-year olds in The Gambia, 5- to 17-month-olds in Burkina Faso, and 5- to 12-month-olds and 10-week-olds in The Gambia. We assessed induction of cellular immunity, taking into account the distinctive hematological status of young infants, and characterized the antibody response to vaccination. T cell responses peaked 7 days after boosting with modified vaccinia virus Ankara (MVA), with highest responses in infants aged 10 weeks at priming. Incorporating lymphocyte count into the calculation of T cell responses facilitated a more physiologically relevant comparison of cellular immunity across different age groups. Both CD8 +  and CD4 + T cells secreted cytokines. Induced antibodies were up to 20-fold higher in all groups compared with Gambian and United Kingdom (UK) adults, with comparable or higher avidity. This immunization regimen elicited strong immune responses, particularly in young infants, supporting future evaluation of efficacy in this key target age group for a malaria vaccine., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Quantifying gametocyte densities in natural malaria infections is important to estimate malaria transmission potential. Two molecular methods (Pfs25 mRNA quantitative reverse transcriptase PCR (qRT-PCR) and Pfs25 mRNA quantitative nucleic acid sequence based amplification (QT-NASBA)) are commonly used to determine gametocyte densities in clinical and epidemiological studies and allow gametocyte detection at densities below the microscopic threshold for detection. Here, reproducibility of these measurements and the association between estimated gametocyte densities and mosquito infection rates were compared., Methods: To quantify intra- and inter-assay variation of QT-NASBA and qRT-PCR, a series of experiments was performed using culture-derived mature Plasmodium falciparum gametocytes from three different parasite isolates (NF54, NF135, NF166). Pfs25 mRNA levels were also determined in samples from clinical trials in Mali and Burkina Faso using both methods. Agreement between the two methods and association with mosquito infection rates in membrane feeding assays were assessed., Results: Intra- and inter-assay variability was larger in QT-NASBA compared to qRT-PCR, particularly at low gametocyte densities (< 1 gametocyte per μL). Logistic models, including log-transformed gametocytaemia estimated by QT-NASBA, explained variability in mosquito feeding experiment results as well as log-transformed gametocytaemia by qRT-PCR (marginal R 2 0.28 and 0.22, respectively). Densities determined by both methods strongly correlated with mosquito infection rates [Spearman's rank correlation coefficient, 0.59 for qRT-PCR and 0.64 for QT-NASBA (P < 0.001 for both)]. Gametocyte densities estimated by qRT-PCR were higher than levels estimated by QT-NASBA or light microscopy at high densities (>100 gametocyte per μL). Samples collected in one of the two transmission studies had extremely low gametocyte densities by both molecular methods, which is suggestive of RNA degradation due to an unknown number of freeze-thaw cycles and illustrates the reliance of molecular gametocyte diagnostics on a reliable cold-chain., Conclusions: The experiments indicate that both qRT-PCR and QT-NASBA are of value for quantifying mature gametocytes in samples collected in field studies. For both assays, estimated gametocyte densities correlated well with mosquito infection rates. QT-NASBA is less reproducible than qRT-PCR, particularly for low gametocyte densities.

Malaria remains a significant global health burden and a vaccine would make a substantial contribution to malaria control. Chimpanzee Adenovirus 63 Modified Vaccinia Ankara Multiple epitope thrombospondin adhesion protein (ME-TRAP) and vaccination has shown significant efficacy against malaria sporozoite challenge in malaria-naive European volunteers and against malaria infection in Kenyan adults. Infants are the target age group for malaria vaccination; however, no studies have yet assessed T-cell responses in children and infants. We enrolled 138 Gambian and Burkinabe children in four different age-groups: 2-6 years old in The Gambia; 5-17 months old in Burkina Faso; 5-12 months old, and also 10 weeks old, in The Gambia; and evaluated the safety and immunogenicity of Chimpanzee Adenovirus 63 Modified Vaccinia Ankara ME-TRAP heterologous prime-boost immunization. The vaccines were well tolerated in all age groups with no vaccine-related serious adverse events. T-cell responses to vaccination peaked 7 days after boosting with Modified Vaccinia Ankara, with T-cell responses highest in 10 week-old infants. Heterologous prime-boost immunization with Chimpanzee Adenovirus 63 and Modified Vaccinia Ankara ME-TRAP was well tolerated in infants and children, inducing strong T-cell responses. We identify an approach that induces potent T-cell responses in infants, which may be useful for preventing other infectious diseases requiring cellular immunity., Competing Interests: Statement The following authors have declared that no conflict of interest exists: MOA, CMB, ABT, AD, IN, YJJ, JUA, JNJ, SHH, NAA, CJD, RR, NKV, OL, AML. KLF, BK, PB, EBI, SBS, KB, KJE. AVSH is a named inventor on patent applications on malaria vectored vaccines and immunization regimens. Authors from ReiThera are employees of and/or shareholders in ReiThera, which is developing vectored vaccines for malaria and other diseases.

The antibody-dependent respiratory burst and opsonic phagocytosis assays have been associated with protection against malaria; however, other mechanisms may also be involved. The antibody-dependent cellular inhibition (ADCI) assay is yet to be correlated with protection in longitudinal cohort studies (LCS). We investigated the relationship between ADCI activity of immunoglobulin G before malaria season and risk of malaria in a LCS involving Ghanaian children. High ADCI activity was significantly associated with reduced risk against malaria. Findings here suggest a potential usefulness of the ADCI assay as a correlate of protection to guide malaria vaccine studies.

Background: Unbiased flow cytometry-based methods have become the technique of choice in many laboratories for high-throughput, accurate assessments of malaria parasites in bioassays. A method to quantify live parasites based on mitotracker red CMXRos was recently described but consistent distinction of early ring stages of Plasmodium falciparum from uninfected red blood cells (uRBC) remains a challenge., Methods: Here, a high-throughput, three-parameter (tri-colour) flow cytometry technique based on mitotracker red dye, the nucleic acid dye coriphosphine O (CPO) and the leucocyte marker CD45 for enumerating live parasites in bioassays was developed. The technique was applied to estimate the specific growth inhibition index (SGI) in the antibody-dependent cellular inhibition (ADCI) assay and compared to parasite quantification by microscopy and mitotracker red staining. The Bland-Altman analysis was used to compare biases between SGI estimated by the tri-colour staining technique, mitotracker red and by microscopy., Results: CPO allowed a better separation between early rings and uRBCs compared to mitotracker red resulting in a more accurate estimate of total parasitaemia. The tri-colour technique is rapid, cost effective and robust with comparable sensitivity to microscopy and capable of discriminating between live and dead and/or compromised parasites. Staining for CD45 improved parasitaemia estimates in ADCI assay since high numbers of leucocytes interfered with the accurate identification of parasitized RBC. The least bias (-1.60) in SGI was observed between the tri-colour and microscopy., Conclusion: An improved methodology for high-throughput assessment of P. falciparum parasitaemia under culture conditions that could be useful in different bioassays, including ADCI and growth inhibition assays has been developed.

Prior to a chimpanzee adenovirus-based (ChAd63) malarial vaccine trial, sera were collected to assess ChAd63-specific neutralizing antibody titers in Banfora (Burkina Faso). The low neutralizing antibody titers reported in both adults and children (median titers, 139.1 and 35.0, respectively) are encouraging for the potential use of ChAd63 as a malarial vaccine vector., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Background: The aim of this study was to determine the incidence and seasonal pattern of malaria in children in South-West Burkina Faso, and to compare, in a randomized trial, characteristics of cases detected by active and passive surveillance. This study also enabled the planning of a malaria vaccine trial., Methods: Households with young children, located within 5 kilometers of a health facility, were randomized to one of two malaria surveillance methods. In the first group, children were monitored actively. Each child was visited twice weekly; tympanic temperature was measured, and if the child had a fever or history of fever, a malaria rapid diagnostic test was performed and a blood smear collected. In the second group, children were monitored passively. The child's parent or caregiver was asked to bring the child to the nearest clinic if he was unwell. Follow up lasted 13 months from September 2009., Results: Incidence of malaria (Fever with parasitaemia ≥5,000/µL) was 1.18 episodes/child/year in the active cohort and 0.89 in the passive cohort (rate ratio 1.32, 95% CI 1.13-1.54). Malaria cases in the passive cohort were more likely to have high grade fever; but parasite densities were similar in the two groups. Incidence was highly seasonal; when a specific case definition was used, about 60% of cases occurred within the 4 months June-September., Conclusion: Passive case detection required at least a 30%-40% increase in the sample size for vaccine trials, compared to active detection, to achieve the same power. However we did not find any evidence that parasite densities were higher with passive than with active detection. The incidence of malaria is highly seasonal and meets the WHO criteria for Seasonal Malaria Chemoprevention (SMC). At least half of the malaria cases in these children could potentially be prevented if SMC was effectively deployed.

Malaria remains a major public health problem due to the emergence and spread of Plasmodium falciparum drug resistance. There is an urgent need to investigate new sources of antimalarial drugs which are more effective against Plasmodium falciparum. One of the potential sources of antimalarial drugs is traditional medicinal plants. In this work, we studied the in vitro antiplasmodial activity of chloromethylenic, methanolic, and MeOH/H2O (1/1) crude extracts and decoction obtained from eight medicinal plants collected in Burkina Faso and of total alkaloids for five plants. Extracts were evaluated in vitro for efficacy against Plasmodium falciparum strain K1, which is resistant to chloroquine, pyrimethamine and proguanil using the fluorescence-based SYBR Green I assay. The antiproliferative activity on human-derived hepatoma cell line HepG2 and Chinese hamster ovary (CHO) cells was evaluated using the 3-[4,5-dimethylthyazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test in order to determine the selectivity index. Among the plant extracts tested for in vitro antiplasmodial activity, 16 were considered to be inactive (with IC50 > 10 μg/ml), six showed a moderate activity (5 < IC50 ≤ 10 μg/ml), and six were found to have a good in vitro activity with IC50 value ≤ 5 μg/ml. The highest antiplasmodial activity was found for extracts from: the alkaloid leaf extract and the chloromethylenic extracts of Combretum fragrans (IC50 = 3 μg/ml, IC50 = 5 μg/ml), the total alkaloids and the chloromethylenic leaf extracts of Combretum collinum (IC50 = 4 μg/ml), the MeOH/H2O leaf extract of Terminalia avicennioides (IC50 = 3.5 μg/ml), and the alkaloid leaf extract of Pavetta crassipes (IC50 = 5 μg/ml). Three other extracts showed moderate antiplasmodial activity (5 < IC50 ≤ 10 μg/ml): Terminalia avicennioides and Combretum fragrans methanolic extracts and Acacia kirkii alkaloid leaf extract (IC50 = 6.5, 9 and 10 μg/ml respectively). The Terminalia avicennioides crude MeOH/H2O (80:20 v/v) extract of the leaves was submitted to a successive liquid/liquid extraction with ethylacetate and n-butanol respectively. The extracts were investigated for in vitro antiplasmodial activity and antioxidant properties using DPPH(·), ABTS(+) and FRAP methods. The ethylacetate extract showed the best antiplasmodial activity (7 μg/ml) and the active constituent was isolated as ellagic acid by bioguided fractionation with an IC50 = 0.2 μM on Plasmodium falciparum and SI = 152. Besides, Terminalia avicennioides leaf extract and ellagic acid showed a good antioxidant activity. Our finding confirms the importance of investigating the antimalarial activity of plant species used in traditional medicine. Overall, two plants belonging to the Combretaceae family, Combretum fragrans and Combretum collinum appeared to be the best candidates and will be further investigated for their antiplasmodial properties, in order to isolate the molecules responsible for the antiplasmodial activity.

Background: In malaria-endemic countries, large proportions of individuals infected with Plasmodium falciparum are asymptomatic and constitute a reservoir of parasites for infection of newly hatched mosquitoes., Methods: Two studies were run in parallel in Burkina Faso to evaluate the impact of systematic identification and treatment of asymptomatic carriers of P. falciparum, detected by rapid diagnostic test, on disease transmission and susceptibility to clinical malaria episodes. A clinical study assessed the incidence of symptomatic malaria episodes with a parasite density >5,000/μL after three screening and treatment campaigns ~1 month apart before the rainy season; and an entomological study determined the effect of these campaigns on malaria transmission as measured by entomological inoculation rate., Results: The intervention arm had lower prevalence of asymptomatic carriers of asexual parasites and lower prevalence of gametocyte carriers during campaigns 2 and 3 as compared to the control arm. During the entire follow-up period, out of 13,767 at-risk subjects, 2,516 subjects (intervention arm 1,332; control arm 1,184) had symptomatic malaria. Kaplan-Meier analysis of the incidence of first symptomatic malaria episode with a parasite density >5,000/μL showed that, in the total population, the two treatment arms were similar until Week 11-12 after campaign 3, corresponding with the beginning of the malaria transmission season, after which the probability of being free of symptomatic malaria was lower in the intervention arm (logrank p < 0.0001). Similar trends were observed in infants and children <5 years and in individuals ≥5 years of age. In infants and children <5 years old who experienced symptomatic malaria episodes, the geometric mean P. falciparum density was lower in the intervention arm than the control arm. This trend was not seen in those individuals aged ≥5 years. Over the year, monthly variation in mosquito density and entomological inoculation rate was comparable in both arms, with September peaks in both indices., Conclusion: Community screening and targeted treatment of asymptomatic carriers of P. falciparum had no effect on the dynamics of malaria transmission, but seemed to be associated with an increase in the treated community's susceptibility to symptomatic malaria episodes after the screening campaigns had finished. These results highlight the importance of further exploratory studies to better understand the dynamics of disease transmission in the context of malaria elimination.

Introduction: Parasitological confirmation before administration of antimalarial treatment has been recommended by the World Health Organization in everyone presenting with symptoms suggestive of malaria at all levels of the health system., Methods: The authors assessed the performance of a histidine-rich protein 2-based malaria rapid diagnostic test used by community health workers in the context of an integrated approach to diagnosis and treatment for malaria and pneumonia. A total of 525 children below 5 years of age were recruited into the study. Children with fever/history of fever within the last 24 h were tested with the rapid diagnostic test (RDT) and a blood smear was obtained for delayed reading., Results: Overall, the FirstSign™ Malaria Pf (Unimed International Inc, South San Francisco, USA) has shown a high sensitivity profile of 97.9% (95% CI 96.3-98.8), but a low specificity of 53.4% (95% CI 49.1-57.7). The specificity was significantly lower during the high transmission season at 25.4% (95% CI 20.5-31.0) compared to 63.7% (95% CI 57.6-69.4%) at the low transmission season. The negative predictive value (NPV) was 95.4% (95% CI 93.2-96.9) and positive predictive value was 71.7% (95% CI 67.7-75.4). The NPV was significantly higher during the low transmission season at 98.2% (95% CI 95.7-99.3) than compared to 80.0% (95% CI 74.7-84.4) at the high transmission season., Conclusion: With such a low specificity, caution should be exercised when using these RDTs for community case management of malaria.

Malaria congenital infection constitutes a major risk in malaria endemic areas. In this study, we report the prevalence of transplacental malaria in Burkina Faso. In labour and delivery units, thick and thin blood films were made from maternal, placental, and umbilical cord blood to determine malaria infection. A total of 1,309 mother/baby pairs were recruited. Eighteen cord blood samples (1.4%) contained malaria parasites (Plasmodium falciparum). Out of the 369 (28.2%) women with peripheral positive parasitemia, 211 (57.2%) had placental malaria and 14 (3.8%) had malaria parasites in their umbilical cord blood. The umbilical cord parasitemia levels were statistically associated with the presence of maternal peripheral parasitemia (OR = 9.24, P ≪ 0.001), placental parasitemia (OR = 10.74, P ≪ 0.001), high-density peripheral parasitemia (OR = 9.62, P ≪ 0.001), and high-density placental parasitemia (OR = 4.91, P = 0.03). In Burkina Faso, the mother-to-child transmission rate of malaria appears to be low.

To determine the relationship between malaria transmission intensity, clinical malaria, immune response, plasmodic index, and to furthermore characterize a malaria vaccine trial site for potential malaria vaccines candidate testing, a study was conducted in Tensobtenga and Balonguen, two villages in Burkina Faso characterized by different malaria transmission levels. The study villages are located in a Sudan savanna area. Malaria transmission is seasonal and peaks in September in these villages. Tensobtenga and Balonguen are comparables in all aspects, except the presence of an artificial lake and wetlands in Tensobtenga. The mosquitoes sampling sites were randomly selected, taking into consideration the number of potential breeding sites, and the number of households in each village. Three times a week during 12 mo mosquitoes were collected by the Center for Disease Control and Prevention light traps in sentinel sites. To assess the infectivity the mosquitoes double ELISAs tests were performed on thoraces of female Anopheles gambiae s.l. (Giles) and Anopheles funestus. A total of 54,392 female Anopheles, representing 92.71% of the total mosquitoes, were collected. The peaks of aggressiveness because of either An. gambiae s.l. or An. funestus were observed in September in each of the villages. However, these peaks were lower in Balonguen compared with Tensobtenga. Malaria cumulative aggressiveness and transmission intensity because of both species peaked in September in each of the two villages, with lower values in Balonguen in comparison to Tensobtenga From February to May, malaria transmission intensity is negligible in Balonguen and <1 bite/person/mo is observed in Tensobtenga. These results have confirmed the marked seasonality of malaria transmission in the study area.

Background: Man to mosquito transmission of malaria depends on the presence of the sexual stage parasites, gametocytes, that often circulate at low densities. Gametocyte densities below the microscopical threshold of detection may be sufficient to infect mosquitoes but the importance of submicroscopical gametocyte carriage in different transmission settings is unknown., Methodology/principal Findings: Membrane feeding experiments were carried out on 80 children below 14 years of age at the end of the wet season in an area of seasonal malaria transmission in Burkina Faso. Gametocytes were quantified by microscopy and by Pfs25-based quantitative nucleic acid sequence-based amplification assay (QT-NASBA). The children's infectiousness was determined by membrane feeding experiments in which a venous blood sample was offered to locally reared Anopheles mosquitoes. Gametocytes were detected in 30.0% (24/80) of the children by microscopy compared to 91.6% (65/71) by QT-NASBA (p<0.001). We observed a strong association between QT-NASBA gametocyte density and infection rates (p = 0.007). Children with microscopically detectable gametocytes were more likely to be infectious (68.2% compared to 31.7% of carriers of submicroscopical gametocytes, p = 0.001), and on average infected more mosquitoes (13.2% compared to 2.3%, p<0.001). However, because of the high prevalence of submicroscopical gametocyte carriage in the study population, carriers of sub-microscopical gametocytes were responsible for 24.2% of the malaria transmission in this population., Conclusions/significance: Submicroscopical gametocyte carriage is common in an area of seasonal transmission in Burkina Faso and contributes substantially to the human infectious reservoir. Submicroscopical gametocyte carriage should therefore be considered when implementing interventions that aim to reduce malaria transmission.