Effect of weekly fever-screening and treatment and monthly RDT testing and treatment on the infectious reservoir of malaria parasites in Burkina Faso: a cluster-randomised trial.

BACKGROUND: The majority of Plasmodium spp infections in endemic countries are asymptomatic and a source of onward transmission to mosquitoes. We aimed to examine whether Plasmodium falciparum transmission and malaria burden could be reduced by improving early detection and treatment of infections with active screening approaches. METHODS: In this 18-month cluster randomised study in Sapone, Burkina Faso, households were enrolled and randomly assigned (1:1:1) to one of three groups: group 1 (control) received standard of care only, group 2 received active weekly, at home, fever screening by a community health worker regardless of symptoms, participants with a fever received a rapid diagnostic test (RDT) and treatment if RDT positive, and group 3 received active weekly fever screening (as in group 2) plus a monthly RDT regardless of symptoms, and treatment if RDT positive. Eligible households had a minimum of three eligible residents, one in each age group (<5 years, 5-15 years, and >15 years). The primary outcome was parasite prevalence by quantitative PCR (qPCR) in the end-of-study cross-sectional survey. Secondary outcomes included parasite and gametocyte prevalence and density in all three end-of-season cross-sectional surveys, incidence of infection, and the transmissibility of infections to mosquitoes. This trial was registered at ClinicalTrials.gov (NCT03705624) and is completed. FINDINGS: A total of 906 individuals from 181 households were enrolled during two phases, and participated in the study. 412 individuals were enrolled between Aug 9 and 17, 2018, and participated in phase 1 and 494 individuals were enrolled between Jan 10 and 31, 2019, in phase 2. In the end-of-study cross-sectional survey (conducted between Jan 13 and 21, 2020), Pfalciparum prevalence by qPCR was significantly lower in group 3 (29·26%; 79 of 270), but not in group 2 (45·66%; 121 of 265), when compared with group 1 (48·72%; 133 of 273; risk ratio 0·65 [95% CI 0·52-0·81]; p=0·0001). Total parasite and gametocyte prevalence and density were also significantly lower in group 3 in all surveys. The largest differences were seen at the end of the dry season, with gametocyte prevalence 78·4% and predicted transmission potential 98·2% lower in group 3 than in group 1. INTERPRETATION: Active monthly RDT testing and treatment can reduce parasite carriage and the infectious reservoir of P falciparum to less than 2% when used during the dry season. This insight might inform approaches for malaria control and elimination. FUNDING: Bill & Melinda Gates Foundation, European Research Council, and The Netherlands Organization for Scientific Research.

Understanding and maximising the community impact of seasonal malaria chemoprevention in Burkina Faso (INDIE-SMC): study protocol for a cluster randomised evaluation trial.

INTRODUCTION: Seasonal malaria chemoprevention (SMC) involves repeated administrations of sulfadoxine-pyrimethamine plus amodiaquine to children below the age of 5 years during the peak transmission season in areas of seasonal malaria transmission. While highly impactful in reducing Plasmodium falciparum malaria burden in controlled research settings, the impact of SMC on infection prevalence is moderate in real-life settings. It remains unclear what drives this efficacy decay. Recently, the WHO widened the scope for SMC to target all vulnerable populations. The Ministry of Health (MoH) in Burkina Faso is considering extending SMC to children below 10 years old. We aim to assess the impact of SMC on clinical incidence and parasite prevalence and quantify the human infectious reservoir for malaria in this population. METHODS AND ANALYSIS: We will perform a cluster randomised trial in Saponé Health District, Burkina Faso, with three study arms comprising 62 clusters of three compounds: arm 1 (control): SMC in under 5-year-old children, implemented by the MoH without directly observed treatment (DOT) for the full course of SMC; arm 2 (intervention): SMC in under 5-year-old children, with DOT for the full course of SMC; arm 3 (intervention): SMC in under 10-year-old children, with DOT for the full course of SMC. The primary endpoint is parasite prevalence at the end of the malaria transmission season. Secondary endpoints include the impact of SMC on clinical incidence. Factors affecting SMC uptake, treatment adherence, drug concentrations, parasite resistance markers and transmission of parasites will be determined. ETHICS AND DISSEMINATION: The London School of Hygiene & Tropical Medicine's Ethics Committee (29193) and the Burkina Faso National Medical Ethics Committee (Deliberation No 2023-05-104) approved this study. The findings will be presented to the community; disease occurrence data and study outcomes will also be shared with the Burkina Faso MoH. Findings will be published irrespective of their results. TRIAL REGISTRATION NUMBER: NCT05878366.

Contribution of the Rapid LAMP-Based Diagnostic Test (RLDT) to the Evaluation of Enterotoxigenic Escherichia coli (ETEC) and Shigella in Childhood Diarrhea in the Peri-Urban Area of Ouagadougou, Burkina Faso.

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.

Impact of Plasmodium falciparum infection on DNA methylation of circulating immune cells.

The regulation of immune cell responses to infection is a complex process that involves various molecular mechanisms, including epigenetic regulation. DNA methylation has been shown to play central roles in regulating gene expression and modulating cell response during infection. However, the nature and extent to which DNA methylation is involved in the host immune response in human malaria remains largely unknown. Here, we present a longitudinal study investigating the temporal dynamics of genome-wide in vivo DNA methylation profiles using 189 MethylationEPIC 850 K profiles from 66 children in Burkina Faso, West Africa, sampled three times: before infection, during symptomatic parasitemia, and after malaria treatment. The results revealed major changes in the DNA methylation profiles of children in response to both Plasmodium falciparum infection and malaria treatment, with widespread hypomethylation of CpGs upon infection (82% of 6.8 K differentially methylated regions). We document a remarkable reversal of CpG methylation profiles upon treatment to pre-infection states. These changes implicate divergence in core immune processes, including the regulation of lymphocyte, neutrophil, and myeloid leukocyte function. Integrative DNA methylation-mRNA analysis of a top differentially methylated region overlapping the pro-inflammatory gene TNF implicates DNA methylation of TNF cis regulatory elements in the molecular mechanisms of TNF regulation in human malaria. Our results highlight a central role of epigenetic regulation in mounting the host immune response to P. falciparum infection and in response to malaria treatment.

Assessment of the transmission blocking activity of antimalarial compounds by membrane feeding assays using natural Plasmodium falciparum gametocyte isolates from West-Africa.

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.

Seasonal Malaria Chemoprevention Implementation: Effect on Malaria Incidence and Immunity in a Context of Expansion of P. falciparum Resistant Genotypes with Potential Reduction of the Effectiveness in Sub-Saharan Africa.

Seasonal Malaria Chemoprevention (SMC), which combines amodiaquine (AQ) with sulfadoxine-pyrimethamine (SP), is an effective and promising strategy, recommended by WHO, for controlling malaria morbidity and mortality in areas of intense seasonal transmission. Despite the effectiveness of this strategy, a number of controversies regarding the impact of the development of malaria-specific immunity and challenges of the strategy in the context of increasing and expanding antimalarial drugs resistance but also the limited coverage of the SMC in children make the relevance of the SMC questionable, especially in view of the financial and logistical investments. Indeed, the number of malaria cases in the target group, children under 5 years old, has increased while the implementation of SMC is been extended in several African countries. This ambivalence of the SMC strategy, the increase in the prevalence of malaria cases suggests the need to evaluate the SMC and understand some of the factors that may hinder the success of this strategy in the implementation areas. The present review discusses the impact of the SMC on malaria morbidity, parasite resistance to antimalarial drugs, molecular and the immunity affecting the incidence of malaria in children. This approach will contribute to improving the malaria control strategy in highly seasonal transmission areas where the SMC is implemented.

Metabolome modulation of the host adaptive immunity in human malaria.

Host responses to infection with the malaria parasite Plasmodium falciparum vary among individuals for reasons that are poorly understood. Here we reveal metabolic perturbations as a consequence of malaria infection in children and identify an immunosuppressive role of endogenous steroid production in the context of P. falciparum infection. We perform metabolomics on matched samples from children from two ethnic groups in West Africa, before and after infection with seasonal malaria. Analysing 306 global metabolomes, we identify 92 parasitaemia-associated metabolites with impact on the host adaptive immune response. Integrative metabolomic and transcriptomic analyses, and causal mediation and moderation analyses, reveal an infection-driven immunosuppressive role of parasitaemia-associated pregnenolone steroids on lymphocyte function and the expression of key immunoregulatory lymphocyte genes in the Gouin ethnic group. In children from the less malaria-susceptible Fulani ethnic group, we observe opposing responses following infection, consistent with the immunosuppressive role of endogenous steroids in malaria. These findings advance our understanding of P. falciparum pathogenesis in humans and identify potential new targets for antimalarial therapeutic interventions.

Higher gametocyte production and mosquito infectivity in chronic compared to incident Plasmodium falciparum infections.

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.

Integrative genomic analysis reveals mechanisms of immune evasion in P. falciparum malaria.

The mechanisms behind the ability of Plasmodium falciparum to evade host immune system are poorly understood and are a major roadblock in achieving malaria elimination. Here, we use integrative genomic profiling and a longitudinal pediatric cohort in Burkina Faso to demonstrate the role of post-transcriptional regulation in host immune response in malaria. We report a strong signature of miRNA expression differentiation associated with P. falciparum infection (127 out of 320 miRNAs, B-H FDR 5%) and parasitemia (72 miRNAs, B-H FDR 5%). Integrative miRNA-mRNA analysis implicates several infection-responsive miRNAs (e.g., miR-16-5p, miR-15a-5p and miR-181c-5p) promoting lymphocyte cell death. miRNA cis-eQTL analysis using whole-genome sequencing data identified 1,376 genetic variants associated with the expression of 34 miRNAs (B-H FDR 5%). We report a protective effect of rs114136945 minor allele on parasitemia mediated through miR-598-3p expression. These results highlight the impact of post-transcriptional regulation, immune cell death processes and host genetic regulatory control in malaria.

Biology of Plasmodium falciparum gametocyte sex ratio and implications in malaria parasite transmission.

While significant advances have been made in understanding Plasmodium falciparum gametocyte biology and its relationship with malaria parasite transmission, the gametocyte sex ratio contribution to this process still remains a relevant research question. The present review discusses the biology of sex determination in P. falciparum, the underlying host and parasite factors, the sex specific susceptibility to drugs, the effect of sex ratio dynamics on malaria parasite transmission and the development of gametocyte sex specific diagnosis tools. Despite the inherent differences across several studies and approaches, the emerging picture highlights a potentially relevant contribution of the P. falciparum gametocyte sex ratio in the modulation of malaria parasite transmission. The increasing availability of molecular methods to measure gametocyte sex ratio will enable evaluation of important parameters, such as the impact of drug treatment on gametocyte sex ratio in vitro and in vivo as well as the changes of gametocyte sex ratios in natural infections, key steps towards elucidating how these parameters affect parasite infectiousness to the mosquito vectors.

Variation in natural exposure to anopheles mosquitoes and its effects on malaria transmission.

Variation in biting frequency by Anopheles mosquitoes can explain some of the heterogeneity in malaria transmission in endemic areas. In this study in Burkina Faso, we assessed natural exposure to mosquitoes by matching the genotype of blood meals from 1066 mosquitoes with blood from residents of local households. We observed that the distribution of mosquito bites exceeded the Pareto rule (20/80) in two of the three surveys performed (20/85, 76, and 96) and, at its most pronounced, is estimated to have profound epidemiological consequences, inflating the basic reproduction number of malaria by 8-fold. The distribution of bites from sporozoite-positive mosquitoes followed a similar pattern, with a small number of individuals within households receiving multiple potentially infectious bites over the period of a few days. Together, our findings indicate that heterogeneity in mosquito exposure contributes considerably to heterogeneity in infection risk and suggest significant variation in malaria transmission potential.

Molecular Characterization of Diarrheagenic Escherichia Coli in Children Less Than 5 Years of Age with Diarrhea in Ouagadougou, Burkina Faso.

Diarrheagenic Escherichia coli (DEC) is important bacteria of children's endemic and epidemic diarrhea worldwide. The aim of this study was to determine the prevalence of DEC isolated from stool samples collected from children with acute diarrhea living in Ouagadougou, Burkina Faso. From August 2013 to October 2015, stool samples were collected from 315 children under 5 years of age suffering from diarrhea in the "Centre Médical avec Antenne Chirurgicale (CMA)" Paul VI and the CMA of Schiphra. E. coli were isolated and identified by standard microbiological methods, and the 16-plex PCR method was used to further characterize them. Four hundred and nineteen (419) E. coli strains were characterized, of which 31 (7.4%) DEC pathotypes were identified and classified in five E. coli pathotypes: 15 enteroaggregative E. coli (EAEC) (48.4%), 8 enteropathogenic E. coli (EPEC) (25.8%) with 4 typical EPEC and 4 atypical EPEC, 4 enteroinvasive E. coli (EIEC) (12.9%), 3 enterohemorrhagic E. coli (EHEC) 9.67%, and 1 enterotoxigenic E. coli (ETEC) 3.2%. The use of multiplex PCR as a routine in clinical laboratory for the detection of DEC would be a useful mean for a rapid management of an acute diarrhea in children.

Clinical Variation of Plasmodium falciparum eba-175, ama-1, and msp-3 Genotypes in Young Children Living in a Seasonally High Malaria Transmission Setting in Burkina Faso.

The association between P. falciparum eba-175, ama-1, and msp-3 polymorphism in the pathogenicity of malaria disease was investigated. We therefore compared the prevalence of different alleles between symptomatic and asymptomatic malarial children under five years of age living in Burkina Faso. Blood filter papers were collected during the 2008 malaria transmission season from 228 symptomatic and 199 asymptomatic children under five years of age. All patients were living in the rural area of Saponé at about 50 km from Ouagadougou, the capital city of Burkina Faso. P. falciparum parasite DNA was extracted using QIAGEN kits and the alleles diversity was assessed by a nested PCR. PCR products were then digested by restriction enzymes based on already described polymorphic regions of the eba-175, ama-1, and msp-3 genes. The individual alleles eba-175_FCR3 and msp-3_K1 frequencies were statistically higher (p < 0.0001) in the asymptomatic group compared to the symptomatic ones. No statistically significant difference was noted in the prevalence of ama-1-3D7, ama-1-K1, and ama-1-HB3 genotypes between the two groups (p > 0.05). The comparative analysis of P. falciparum genotypes indicated that the polymorphism in eba-175 and msp-3 genotypes varied between asymptomatic and symptomatic clinical groups and may contribute to the pathogenesis of malaria.