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Background Long-lasting insecticidal nets (LLINs) are currently the primary method of malaria control in sub-Saharan Africa and have contributed to a significant reduction in malaria burden over the past 15 years. However, this progress is threatened by the wide-scale selection of insecticide-resistant malaria vectors. It is, therefore, important to accelerate the generation of evidence for new classes of LLINs. Methods This protocol presents a three-arm superiority, single-blinded, cluster randomized controlled trial to evaluate the impact of 2 novel dual-active ingredient LLINs on epidemiological and entomological outcomes in Benin, a malaria-endemic area with highly pyrethroid-resistant vector populations. The study arms consist of (i) Royal Guard® LLIN, a net combining a pyrethroid (alpha-cypermethrin) plus an insect growth regulator (pyriproxyfen), which in the adult female is known to disrupt reproduction and egg fertility; (ii) Interceptor G2® LLIN, a net incorporating two adulticides (alpha-cypermethrin and chlorfenapyr) with different modes of action; and (iii) the control arm, Interceptor® LLIN, a pyrethroid (alpha-cypermethrin) only LLIN. In all arms, one net for every 2 people will be distributed to each household. Sixty clusters were identified and randomised 1:1:1 to each study arm. The primary outcome is malaria case incidence measured over 24 months through active case detection in a cohort of 25 children aged 6 months to 10 years, randomly selected from each cluster. Secondary outcomes include 1) malaria infection prevalence (all ages) and prevalence of moderate to severe anaemia in children under 5 years old, measured at 6 and 18 months post-intervention; 2) entomological indices measured every 3 months using human landing catches over 24 months. Insecticide resistance intensity will also be monitored over the study period. Discussion This study is the second cluster randomised controlled trial to evaluate the efficacy of these next-generation LLINs to control malaria transmitted by insecticide-resistant mosquitoes. The results of this study will form part of the WHO evidence-based review to support potential public health recommendations of these nets and shape malaria control strategies of sub-Saharan Africa for the next decade. Trial registration ClinicalTrials.gov, NCT03931473, registered on 30 April 2019.

Background This study provides detailed characteristics of vector populations in preparation for a three-arm cluster randomized controlled trial (RCT) aiming to compare the community impact of dual active-ingredient (AI) long-lasting insecticidal nets (LLINs) that combine two novel insecticide classes–chlorfenapyr or pyriproxifen–with alpha-cypermethrin to improve the prevention of malaria transmitted by insecticide-resistant vectors compared to standard pyrethroid LLINs. Methods The study was carried out in 60 villages across Cove, Zangnanando and Ouinhi districts, southern Benin. Mosquito collections were performed using human landing catches (HLCs). After morphological identification, a sub-sample of Anopheles gambiae s.l. were dissected for parity, analyzed by PCR for species and presence of L1014F kdr mutation and by ELISA-CSP to identify Plasmodium falciparum sporozoite infection. WHO susceptibility tube tests were performed by exposing adult An. gambiae s.l., collected as larvae from each district, to 0.05% alphacypermethrin, 0.75% permethrin, 0.1% bendiocarb and 0.25% pirimiphos-methyl. Synergist assays were also conducted with exposure first to 4% PBO followed by alpha-cypermethrin. Results An. gambiae s.l. (n = 10807) was the main malaria vector complex found followed by Anopheles funestus s.l. (n = 397) and Anopheles nili (n = 82). An. gambiae s.l. was comprised of An. coluzzii (53.9%) and An. gambiae s.s. (46.1%), both displaying a frequency of the L1014F kdr mutation >80%. Although more than 80% of people slept under standard LLIN, human biting rate (HBR) in An. gambiae s.l. was higher indoors [26.5 bite/person/night (95% CI: 25.2–27.9)] than outdoors [18.5 b/p/n (95% CI: 17.4–19.6)], as were the trends for sporozoite rate (SR) [2.9% (95% CI: 1.7–4.8) vs 1.8% (95% CI: 0.6–3.8)] and entomological inoculation rate (EIR) [21.6 infected bites/person/month (95% CI: 20.4–22.8) vs 5.4 (95% CI: 4.8–6.0)]. Parous rate was 81.6% (95%CI: 75.4–88.4). An. gambiae s.l. was resistant to alpha-cypermethrin and permethrin but, fully susceptible to bendiocarb and pirimiphos-methyl. PBO pre-exposure followed by alpha-cypermethrin treatment induced a higher 24 hours mortality compared to alphacypermethrin alone but not exceeding 40%. Conclusions Despite a high usage of standard pyrethroid LLINs, the study area is characterized by intense malaria transmission. The main vectors An. coluzzii and An. gambiae s.s. were both highly resistant to pyrethroids and displayed multiple resistance mechanisms, L1014F kdr mutation and mixed function oxidases. These conditions of the study area make it an appropriate site to conduct the trial that aims to assess the effect of novel dual-AI LLINs on malaria transmitted by insecticide-resistant vectors.

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Malaria remains the main cause of morbidity and mortality in Benin despite the scale-up of long-lasting insecticidal nets (LLINs), indoor residual spraying, and malaria case management. This study aimed to determine the malaria burden and its associated risk factors in a rural area of Benin characterized by high net coverage and pyrethroid-resistant mosquito vectors. A community-based cross-sectional survey was conducted in three districts in southern Benin. Approximately 4,320 randomly selected participants of all ages were tested for malaria using rapid diagnostic tests within 60 clusters. Risk factors for malaria infection were evaluated using mixed-effect logistic regression models. Despite high population net use (96%), malaria infection prevalence was 43.5% (cluster range: 15.1–72.7%). Children (58.7%) were more likely to be infected than adults (31.2%), with a higher malaria prevalence among older children (5–10 years: 69.1%; 10–15 years: 67.9%) compared with young children (< 5 years: 42.1%); however, young children were more likely to be symptomatic. High household density, low socioeconomic status, young age (< 15 years), poor net conditions, and low net usage during the previous week were significantly associated with malaria infection. Malaria prevalence remains high in this area of intense pyrethroid resistance despite high net use. New classes of LLINs effective against resistant vectors are therefore crucial to further reduce malaria in this area.

In malaria-endemic areas, subjects from specific groups like Fulani have a peculiar protection against malaria, with high levels of IgM but also frequent anemia and splenomegaly. The mechanisms underlying this phenotype remain elusive. In Benin, West Africa, we measured the deformability of circulating erythrocytes in genetically distinct groups (including Fulani) living in sympatry, using ektacytometry and microsphiltration, a mimic of how the spleen clears rigid erythrocytes. Compared to non-Fulani, Fulani displayed a higher deformability of circulating erythrocytes, pointing to an enhanced clearance of rigid erythrocytes by the spleen. This phenotype was observed in individuals displaying markers of Plasmodium falciparum infection. The heritability of this new trait was high, with a strong multigenic component. Five of the top 10 genes selected by a population structure-adjusted GWAS, expressed in the spleen, are potentially involved in splenic clearance of erythrocytes (CHERP, MB, PALLD, SPARC, PDE10A), through control of vascular tone, collagen synthesis and macrophage activity. In specific ethnic groups, genetically-controlled processes likely enhance the innate retention of infected and uninfected erythrocytes in the spleen, explaining splenomegaly, anemia, cryptic intrasplenic parasite loads, hyper-IgM, and partial protection against malaria. Beyond malaria-related phenotypes, inherited splenic hyper-filtration of erythrocytes may impact the pathogenesis of other hematologic diseases.Research in contextEvidence before this studyThe genetic background of individuals influences their susceptibility to infectious diseases. Specific human groups, like the Fulani in Africa, react to malaria parasites (named Plasmodium) in a specific way. Upon infection, Fulani develop a grossly enlarged spleen, and high levels of anti-Plasmodium antibodies in their blood. They also carry smaller numbers of parasites in their blood, and thus are considered partially protected against malaria. The mechanisms underlying this natural protection, different from other natural protective mechanisms such as the sickle cell trait, are not well understood.Malaria impairs the deformability of red blood cells and the spleen is a key organ to controlling red blood cell quality. We have recently demonstrated that red blood cells containing live malaria parasites accumulate intensely in the spleen of subjects with long term exposure to these parasites. Enhanced retention of infected and uninfected red blood cells in the spleen would explain why the spleen is larger and why lower numbers of parasites are left in circulation. We thus explored whether the retention of infected and uninfected red blood cells could explain why Fulani are partially protected against malaria. Because it is unethical to perform spleen puncture or biopsies for research purposes, our explorations were indirect by carefully analyzing the properties of circulating red blood cells in a large number of subjects and by assessing whether observations could be explained by their genetic make-up.Added value of this studyIn more than 500 subjects, we confirmed the high frequency of large spleens in Fulani and, through 2 different methods, we demonstrated an enhanced deformability of their circulating red blood cells, that likely stems from the more efficient removal of the less deformable ones. This enhanced deformability was found to be inheritable based on carefully collected family links and refined analysis of genetic markers.Implications of all the available evidenceOur findings indicate that genes potentially driving the filtration of red blood cells by the spleen likely influence how subjects in specific groups in Africa and elsewhere react to malaria. While most previous hypotheses pointed to conventional immunological mechanisms as the trigger, we propose that a simple physiological mechanism that controls the quality of red blood cells may drive natural protection from malaria even before the intervention of immunological cells. A better understanding of these processes is of great importance in the context of malaria elimination efforts.These findings may also have an impact on the understanding of other red blood cell-related disorders, such as inherited red cell diseases, in which splenic filtration of abnormal red blood cells may precipitate splenic complications.

Introduction: malaria is one of the most frequent hematological diseases worldwide. Because the malaria parasite Plasmodium falciparum develops mainly in red blood cells (RBC), splenic retention of infected and uninfected RBC is likely a key player in the variable susceptibility of humans to malaria. Age and ethnicity are important determinants of the manifestations of malaria in Africa (Reyburn JAMA 2005, Dolo Am J Trop Med Hyg 2005; Greenwood Ann Trop Med Parasitol 1987; Torcia PNAS 2008), Asia (Price Am J Trop Med Hyg 2001), and in travelers (Seringe Emerg Infect Dis 2011). We had speculated that variations in the splenic sensing of RBC contribute to the innate protection/susceptibility of infants against distinct forms of severe malaria and to the pathogenesis of chronic malaria (Buffet Curr Opin Hematol 2009; Buffet Blood 2011). Here, we explore the deformability and morphology of circulating RBC in populations living in a malaria-endemic area. Materials and methods: experiments were embedded in an integrated study driven by Institut de Recherche pour le Développement, which aims at the identification of genetic, epidemiologic and anthropologic determinants of susceptibility to malaria. IRB approval was obtained from Institut des Sciences Biomédicales Appliquées, Benin. Clinical and biological data were collected at the beginning of the rainy season from 627 individuals, belonging to 4 different ethnic groups living in sympatry in Atakora, North Benin and included age, gender, ethnicity, body temperature, presence and grade of splenomegaly, rapid diagnostic test for malaria (RDT), thick film and rapid hemoglobin determination with HemoCue©. Venous blood was collected for determination of RBC morphology and deformability. Using microsphiltration, a RBC filtering method that uses microsphere layers to mimic the mechanical retention of RBC in the splenic red pulp (Deplaine Blood 2011) we quantified the ability of a mix of labeled and non-labeled RBCs to squeeze between calibrated slits, results being expressed as retention or enrichment rates (RER) of subject's RBC compared to normal RBC (from a single French O-positive donor) stored in blood bank conditions. Microsphiltration has been adapted to high-throughput experimentation using microplates (Duez AAC 2015). Experiments were performed in a field laboratory established on site; microplates were prepared in Paris and brought to North Benin in luggage with constant care to avoid shocks during transportation. All RBC samples were filtered in triplicate less than 8 hours after blood collection. Up- and downstream samples were brought back to France at 4°C in sealed micro-well plates and analyzed for individual RER calculation in the next 2 weeks by flow cytometry. Results: over 10 days, 262 adults and 249 children were included, 31% Bariba, 17% Gando (genetically related to Bariba), 24% Otamari, 27% Peulhs. Prevalences of splenomegaly, positive RDT, and fever were 13%, 27%, and 2%, respectively. Of 629 blood samples collected, 511 could be analyzed. RER of controls remained stable with time and across 17 microfiltering plates, with a median (IQR) retention rate of 12% (5% - 21%). Ethnicity and age were the only two factors associated with statistically significant differences in RER (figures 1 and 2). Infants (less than 2 year-old) had a more important enrichment than older children and adults (median in 2 years old or less 287%; 3 to 5 years 103%; 6 to 10 years: 64%; more than ten years: 91%; p=0.0161). Peulhs and Otamari also had higher median enrichment rates than Bariba and Gando (RER: 122% and 118%, 75%, 64%, respectively; p=0.0246). Conversely, splenomegaly, gender, positivity of RDT or anemia at the time of sampling were not associated with RER. Discussion: higher averaged enrichment rates in specific ethnic subgroups, namely Peulhs and Otamari, likely result from a more stringent splenic retention, leaving more deformable RBC in circulation. An innate spleen-RBC interaction process was also observed in infants, which is consistent with the higher incidence of severe malarial anemia and splenomegaly observed in this population (Reyburn JAMA 2005; Price Am J Trop Med Hyg 2001). Our results show that innate factors (e.g. ethnicity and age) tend to influence the deformability of RBC, and therefore the phenotypic expression of malaria in Africa. Ongoing experiments aim at deciphering the mechanisms responsible for these differences. Disclosures No relevant conflicts of interest to declare.

Malaria remains the main cause of morbidity and mortality in Benin despite the scale-up of long-lasting insecticidal nets (LLINs), indoor residual spraying, and malaria case management. This study aimed to determine the malaria burden and its associated risk factors in a rural area of Benin characterized by high net coverage and pyrethroid-resistant mosquito vectors. A community-based cross-sectional survey was conducted in three districts in southern Benin. Approximately 4,320 randomly selected participants of all ages were tested for malaria using rapid diagnostic tests within 60 clusters. Risk factors for malaria infection were evaluated using mixed-effect logistic regression models. Despite high population net use (96%), malaria infection prevalence was 43.5% (cluster range: 15.1-72.7%). Children (58.7%) were more likely to be infected than adults (31.2%), with a higher malaria prevalence among older children (5-10 years: 69.1%; 10-15 years: 67.9%) compared to young children (<5 years: 42.1%); however, young children were more likely to be symptomatic. High household density, low socioeconomic status, young age (<15years), poor net conditions, and low net usage during the previous week were significantly associated with malaria infection. Malaria prevalence remains high in this area of intense pyrethroid resistance despite high net use. New classes of LLINs effective against resistant vectors are therefore crucial to further reduce malaria in this area.