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Despite its epidemiological importance, the time Plasmodium parasites take to achieve development in the vector mosquito (the extrinsic incubation period, EIP) remains poorly characterized. A novel non-destructive assay designed to estimate EIP in single mosquitoes, and more broadly to study Plasmodium–Anopheles vectors interactions, is presented. The assay uses small pieces of cotton wool soaked in sugar solution to collect malaria sporozoites from individual mosquitoes during sugar feeding to monitor infection status over time. This technique has been tested across four natural malaria mosquito species of Africa and Asia, infected with Plasmodium falciparum (six field isolates from gametocyte-infected patients in Burkina Faso and the NF54 strain) and across a range of temperatures relevant to malaria transmission in field conditions. Monitoring individual infectious mosquitoes was feasible. The estimated median EIP of P. falciparum at 27 °C was 11 to 14 days depending on mosquito species and parasite isolate. Long-term individual tracking revealed that sporozoites transfer onto cotton wool can occur at least until day 40 post-infection. Short individual EIP were associated with short mosquito lifespan. Correlations between mosquito/parasite traits often reveal trade-offs and constraints and have important implications for understanding the evolution of parasite transmission strategies.

Background Mass drug administration (MDA) with azithromycin (AZ) is being considered as a strategy to promote child survival in sub-Saharan Africa, but the mechanism by which AZ reduces mortality is unclear. To better understand the nature and extent of protection provided by AZ, we explored the profile of protection by time since administration, using data from a household-randomized, placebo-controlled trial in Burkina Faso and Mali. Methods Between 2014 and 2016, 30 977 children aged 3–59 months received seasonal malaria chemoprevention (SMC) with sulfadoxine-pyrimethamine plus amodiaquine and either AZ or placebo monthly, on 4 occasions each year. Poisson regression with gamma-distributed random effects, accounting for the household randomization and within-individual clustering of illness episodes, was used to compare incidence of prespecified outcomes between SMC+AZ versus SMC+placebo groups in fixed time strata post-treatment. The likelihood ratio test was used to assess evidence for a time-treatment group interaction. Results Relative to SMC+placebo, there was no evidence of protection from SMC+AZ against hospital admissions and deaths. Additional protection from SMC+AZ against malaria was confined to the first 2 weeks post-administration (protective efficacy (PE): 24.2% [95% CI: 17.8%, 30.1%]). Gastroenteritis and pneumonia were reduced by 29.9% [21.7; 37.3%], and 34.3% [14.9; 49.3%], respectively, in the first 2 weeks postadministration. Protection against nonmalaria fevers with a skin condition persisted up to 28 days: PE: 46.3% [35.1; 55.6%]. Conclusions The benefits of AZ-MDA are broad-ranging but short-lived. To maximize impact, timing of AZ-MDA must address the challenge of targeting asynchronous morbidity and mortality peaks from different causes., Mass administration of azithromycin (AZ-MDA) alongside seasonal malaria chemoprevention in children in Burkina Faso and Mali has broad-ranging but short-lived benefits. To maximize impact, AZ-MDA must address the challenge of targeting asynchronous peaks in morbidity and mortality from different causes.

BackgroundThe use of pyronaridine-artesunate (PA) has been associated with scarce transaminitis in patients. This analysis aimed to evaluate the hepatic safety profile of repeated treatment with PA versus artemether–lumefantrine (AL) in patients with consecutive uncomplicated malaria episodes in Bobo-Dioulasso, Burkina Faso.MethodsThis study analysed data from a clinical trial conducted from 2012 to 2015, in which participants with uncomplicated malaria were assigned to either PA or AL arms and followed up to 42 days. Subsequent malaria episodes within a 2-years follow up period were also treated with the same ACT initially allocated. Transaminases (AST/ALT), alkaline phosphatase (ALP), total and direct bilirubin were measured at days 0 (baseline), 3, 7, 28 and on some unscheduled days if required. The proportions of non-clinical hepatic adverse events (AEs) following first and repeated treatments with PA and AL were compared within study arms. The association of these AEs with retreatment in each arm was also determined using a logistic regression model.ResultsA total of 1379 malaria episodes were included in the intention to treat analysis with 60% of all cases occurring in the AL arm. Overall, 179 non-clinical hepatic AEs were recorded in the AL arm versus 145 in the PA arm. Elevated ALT was noted in 3.05% of treated malaria episodes, elevated AST 3.34%, elevated ALP 1.81%, and elevated total and direct bilirubin in 7.90% and 7.40% respectively. Retreated participants were less likely to experience elevated ALT and AST than first episode treated participants in both arms. One case of Hy’s law condition was recorded in a first treated participant of the PA arm. Participants from the retreatment group were 76% and 84% less likely to have elevated ALT and AST, respectively, in the AL arm and 68% less likely to present elevated ALT in the PA arm. In contrast, they were almost 2 times more likely to experience elevated total bilirubin in both arms.ConclusionsPyronaridine-artesunate and artemether–lumefantrine showed similar hepatic safety when used repeatedly in participants with uncomplicated malaria. Pyronaridine-artesunate represents therefore a suitable alternative to the current first line anti-malarial drugs in use in endemic areas.Trial registrationPan African Clinical Trials Registry. PACTR201105000286876

Mass drug administration (MDA) with azithromycin (AZ) has been used successfully to control trachoma. However, several studies have shown that MDA with AZ has led to the emergence of resistance to AZ in Streptococcus pneumoniae. The emergence of resistance to AZ has also been observed when this antibiotic was combined with the antimalarials used for seasonal malaria chemoprevention (SMC). The development of antibiotic resistance, including resistance to AZ, is sometimes associated with the emergence of a bacterial clone that belongs to a specific serotype. We hypothesize that the increase in resistance of S. pneumoniae observed after 3 years of SMC with AZ might be associated with a change in the distribution of pneumococcal serotypes. Therefore, 698 randomly selected isolates from among the 1,468 isolates of S. pneumoniae obtained during carriage studies undertaken during an SMC plus AZ trial were serotyped. A polymerase chain reaction (PCR) multiplex assay using an algorithm adapted to the detection of the pneumococcal serotypes most prevalent in African countries was used for initial serotyping, and the Quellung technique was used to complement the PCR technique when necessary. Fifty-six serotypes were detected among the 698 isolates of S. pneumoniae. A swift appearance and disappearance of many serotypes was observed, but some serotypes including 6A, 19F, 19A, 23F, and 35B were persistent. The distribution of serotypes between isolates obtained from children who had received AZ or placebo was similar. An increase in AZ resistance was seen in several serotypes following exposure to AZ. Mass drug administration with AZ led to the emergence of resistance in pneumococci of several different serotypes and did not appear to be linked to the emergence of a single serotype.

Background Seasonal malaria chemoprevention (SMC) consists of administration of sulfadoxine-pyrimethamine (SP) + amodiaquine (AQ) at monthly intervals to children during the malaria transmission period. Whether the addition of azithromycin (AZ) to SMC could potentiate the benefit of the intervention was tested through a double-blind, randomized, placebo-controlled trial. The effect of SMC and the addition of AZ, on malaria transmission and on the life history traits of Anopheles gambiae mosquitoes have been investigated. Methods The study included 438 children randomly selected from among participants in the SMC + AZ trial and 198 children from the same area who did not receive chemoprevention. For each participant in the SMC + AZ trial, blood was collected 14 to 21 days post treatment, examined for the presence of malaria sexual and asexual stages and provided as a blood meal to An. gambiae females using a direct membrane-feeding assay. Results The SMC treatment, with or without AZ, significantly reduced the prevalence of asexual Plasmodium falciparum (LRT X22 = 69, P 22 = 54, P 22 = 61, P X22 = 22.8, P 21 = 5.2, P = 0.02), suggesting a significant effect of AZ on gametocyte infectivity. There was a slight negative effect of SPAQ and SPAQ + AZ on mosquito survival compared to mosquitoes fed with blood from control children (LRTX22 = 330, P Conclusion This study demonstrates that SMC may contribute to a reduction in human to mosquito transmission of P. falciparum, and the reduced mosquito longevity observed for females fed on treated blood may increase the benefit of this intervention in control of malaria. The addition of AZ to SPAQ in SMC appeared to enhance the infectivity of gametocytes providing further evidence that this combination is not an appropriate intervention.

Background: Besides feeding on blood, females of the malaria vector Anopheles gambiae s.l. readily feed on natural sources of plant sugars. The impact of toxic secondary phytochemicals contained in plant-derived sugars on mosquito physiology and on the development of Plasmodium parasites remains elusive. The focus of this study was to explore the influence of the alkaloid ricinine, found in the nectar of the castorbean Ricinus communis, on the mosquito ability to transmit Plasmodium falciparum. Methods: Females of Anopheles gambiae and its sibling species Anopheles coluzzii were exposed to ricinine through sugar feeding assays to assess the effect of this phytochemical on mosquito survival, the level of P. falciparum infection, and the growth rate of the parasite. Results: Ricinine induced a significant reduction in the longevity of both Anopheles species. Ricinine caused acceleration in the parasite growth rate with an earlier invasion of the salivary glands in both species. At a concentration of 0.04 g l-1 in An. coluzzii, ricinine had no effect on mosquito infection, while 0.08 g l-1 ricinine-5% glucose solution induced a 14% increase in An. gambiae infection rate. Conclusions: Overall, our findings reveal that consumption of certain nectar phytochemicals can have unsuspected and contrasting effects on key phenotypic traits that govern the intensity of malaria transmission. Further studies will be required before concluding on the putative role of ricinine as a novel control agent, including the development of ricinine-based toxic and transmission-blocking sugar baits. Testing other secondary phytochemicals in plant nectar will provide a broader understanding of the impact, which plants can have on the transmission of vector-borne diseases.

The spread of insecticide resistance in Anopheles mosquitoes and drug resistance in Plasmodium parasites is contributing to a global resurgence of malaria, making the generation of control tools that can overcome these issues an urgent public health priority. We recently showed that the transmission of Plasmodium falciparum parasites can be efficiently blocked when exposing Anopheles gambiae females to antimalarials deposited on a treated surface, with no negative consequences on mosquito fitness. Here, we demonstrate this approach can overcome the hurdles of insecticide resistance in mosquitoes and drug resistant in parasites. We show that the transmission-blocking efficacy of mosquito-targeted antimalarials is maintained when field-derived, insecticide resistant Anopheles are exposed to the potent cytochrome b inhibitor atovaquone, demonstrating that this drug escapes insecticide resistance mechanisms that could potentially interfere with its function. Moreover, this approach prevents transmission of field-derived, artemisinin resistant P. falciparum parasites (Kelch13 C580Y mutant), proving that this strategy could be used to prevent the spread of parasite mutations that induce resistance to front-line antimalarials. Atovaquone is also highly effective at limiting parasite development when ingested by mosquitoes in sugar solutions, including in ongoing infections. These data support the use of mosquito-targeted antimalarials as a promising tool to complement and extend the efficacy of current malaria control interventions.Significance StatementEffective control of malaria is hampered by resistance to vector-targeted insecticides and parasite-targeted drugs. This situation is exacerbated by a critical lack of chemical diversity in both interventions and, as such, new interventions are badly needed. Recent laboratory studies have shown that an alternative approach based on treating Anopheles mosquitoes directly with antimalarial compounds can render the vector incapable of transmitting the Plasmodium parasites that cause malaria. While promising, showing that mosquito-targeted antimalarials remain effective against wild parasites and mosquitoes, including drug- and insecticide-resistant populations, respectively, is crucial to the future viability of this approach. In this study, carried out in the US and Burkina Faso, we show that antimalarial exposure is highly effective, even against extremely resistant mosquitoes, and can block transmission of drug-resistant parasites. By combining lab, and field-based studies in this way we have demonstrated that this novel approach can be effective in areas where conventional control measures are no longer as effective.

Staphylococcus aureus is a major cause of serious illness and death in children, indicating the need to monitor prevalent strains, particularly in the vulnerable pediatric population. Nasal carriage of S. aureus is important as carriers have an increased risk of serious illness due to systemic invasion by this pathogen and can transmit the infection. Recent studies have demonstrated the effectiveness of azithromycin in reducing the prevalence of nasopharyngeal carrying of pneumococci, which are often implicated in respiratory infections in children. However, very few studies of the impact of azithromycin on staphylococci have been undertaken. During a clinical trial under taken in 2016, nasal swabs were collected from 778 children aged 3 to 59 months including 385 children who were swabbed before administration of azithromycin or placebo and 393 after administration of azithromycin or placebo. Azithromycin was given in a dose of 100 mg for three days, together with the antimalarials sulfadoxine-pyrimethamine and amodiaquine, on four occasions at monthly intervals during the malaria transmission season. These samples were cultured for S. aureus as well as for the pneumococcus. The S. aureus isolates were tested for their susceptibility to azithromycin (15 g), penicillin (10 IU), and cefoxitine (30 g) (Oxoid Ltd). S. aureus was isolated from 13.77% (53/385) swabs before administration of azithromycin and from 20.10% (79/393) six months after administration (PR = 1.46 [1.06; 2.01], p = 0.020). Azithromycin resistance found in isolates of S. aureus did not differ significantly before and after intervention (26.42% [14/53] vs 16.46% [13/79], (PR = 0.62 [0.32; 1.23], p = 0.172). Penicillin resistance was very pronounced, 88.68% and 96.20% in pre-intervention and in post-intervention isolates respectively, but very little Methicillin Resistance (MRSA) was detected (2 cases before and 2 cases after intervention). Monitoring antibiotic resistance in S. aureus and other bacteria is especially important in Burkina Faso due to unregulated consumption of antibiotics putting children and others at risk.

Despite its epidemiological importance, the time Plasmodium parasites take to achieve development in the vector mosquito (the extrinsic incubation period, EIP) remains poorly characterized. A novel non-destructive assay designed to estimate EIP in single mosquitoes, and more broadly to study Plasmodium – Anopheles vectors interactions, is presented. The assay uses small pieces of cotton wool soaked in sugar solution to collect malaria sporozoites from individual mosquitoes during sugar feeding to monitor infection status over time. This technique has been tested across four natural malaria mosquito species of Africa and Asia, six parasite isolates of Plasmodium falciparum, and across a range of temperatures relevant to malaria transmission in field conditions. We find that monitoring individual infectious mosquitoes is feasible, although due to the frequency of mosquito sugar feeding and inter-individual variation in infection intensity, there is inherent risk that this technique will result in some false negatives. The sensitivity rate ranged from 0.27 to 0.81 depending on mosquito species and on infection intensity in mosquitoes used to collect saliva. Using this non-destructive technique, the estimated median extrinsic incubation period of P. falciparum at 27°C was 11 to 14 days depending on mosquito species and parasite isolate. Long-term individual tracking also revealed that sporozoite transfer onto cotton wool can occur at least until day 40 post-infection. In addition to contributing to a better understanding of EIP and mosquito to human transmission with implications for improving epidemiological models, this technique also allows to link different transmission traits at the mosquito individual level. As one example, we found a significant relationship between EIP and mosquito lifespan, with short individual EIP associated with short mosquito lifespan. Correlations between mosquito/parasite traits often reveal trade-offs and constraints and have important implications for understanding the evolution of parasite transmission strategies.

Summary The microbiota of Anopheles mosquitoes influences malaria transmission. Antibiotics ingested during a blood meal impact the mosquito microbiome and malaria transmission, with substantial differences between drugs. Here, we assessed if amoxicillin affects the gut mosquito microbiota. We collected Anopheles larvae in Burkina Faso, kept them in semi-field conditions, and offered a blood meal to adult females. We tested the impact of blood supplementation with two alternative amoxicillin preparations on microbiota composition, determined by high-throughput sequencing in individual gut samples. Our analysis detected four major genera, Elizabethkingia, Wigglesworthia, Asaia, and Serratia. The antibiotic treatment significantly affected overall microbiota composition, with a specific decrease in the relative abundance of Elizabethkingia and Asaia during blood digestion. Besides its interest on the influence of amoxicillin on the mosquito microbiota, our study proposes a thorough approach to report negative-control data of high-throughput sequencing studies on samples with a reduced microbial load., Graphical abstract, Highlights • We sequenced bacterial 16S in mosquitoes collected as larvae in Burkina Faso • Elizabethkingia, Wigglesworthia, Asaia, and Serratia dominate the gut microbiota • Amoxicillin treatment and potentially gametocyte presence influence the microbiota • We set up methods to consider mock controls after sequencing low-biomass samples, Entomology; Microbiome

A trial was conducted in Burkina Faso and Mali to investigate whether addition of azithromycin to the antimalarials used for seasonal malaria chemoprevention reduces mortality and hospital admissions of children. We tested the sensitivity of nasal isolates of Streptococcus pneumoniae obtained during this trial to azithromycin and other antibiotics.Azithromycin or placebo was administered monthly, in combination with the antimalarials used for seasonal malaria chemoprevention, for four months, over the annual malaria transmission seasons of 2014, 2015, and 2016. Nasopharyngeal swabs were collected from 2773 Burkinabe and 2709 Malian children on seven occasions: in July and December each year prior to and after drug administration, and at a final survey in early 2018. Pneumococci were isolated from nasopharyngeal swabs and tested for sensitivity to azithromycin and other antibiotics.A total of 5482 samples were collected. In Burkina Faso, the percentage of pneumococcal isolates resistant to azithromycin among children who had received it increased from 4.9% (95% CI: 2.4%, 9.9%) before the intervention to 25.6% (95% CI: 17.6%, 35.7%) afterward. In Mali, the increase was from 7.6% (95% CI: 3.8%, 14.4%) to 68.5% (95% CI: 55.1%, 79.4%). The percentage of resistant isolates remained elevated (17.7% (95% CI: 11.1%, 27.1%) in Burkina Faso and 19.1% (95% CI: 13.5%, 26.3%) in Mali) among children who had received azithromycin 1 year after stopping the intervention. An increase in resistance to azithromycin was also observed in children who had received a placebo but it was less marked.Addition of azithromycin to the antimalarial combination used for seasonal malaria chemoprevention was associated with an increase in resistance of pneumococci to azithromycin and erythromycin, which persisted 1 year after the last administration of azithromycin.Un essai a été mené au Burkina Faso et au Mali pour investiguer si l’addition d’azithromycine aux antipaludéens utilisés dans le cadre de la chimioprévention du paludisme saisonnier réduisait la mortalité et les hospitalisations d’enfants. Nous avons testé la sensibilité à l'azithromycine et à d'autres antibiotiques pour les isolats nasaux de Streptococcus pneumoniae obtenus lors de cet essai. MÉTHODES: L'azithromycine ou un placebo a été administré mensuellement, en association avec les antipaludéens utilisés pour la chimioprévention du paludisme saisonnier, pendant 4 mois, durant les saisons de transmission annuelle du paludisme de 2014, 2015 et 2016. Des échantillons nasopharyngés ont été prélevés sur écouvillons chez 2.773 enfants burkinabés et 2.709 enfants maliens lors de 7 occasions: en juillet et en décembre chaque année avant et après l'administration du médicament, ainsi que lors d'une surveillance finale au début de 2018. Les pneumocoques ont été isolés à partir d'écouvillons nasopharyngés et soumis à des tests de sensibilité à l'azithromycine et à d'autres antibiotiques. RÉSULTATS: 5.482 échantillons ont été collectés. Au Burkina Faso, le pourcentage d’isolats de pneumocoque résistants à l’azithromycine chez les enfants qui l’avaient reçu était passé de 4,9% (IC95%: 2,4%, 9,9%) avant l’intervention à 25,6% (IC95%: 17,6-35,7%) après. Au Mali, l'augmentation est passée de 7,6% (IC95%: 3,8-14,4%) à 68,5% (IC95%: 55,1-79,4%). Le pourcentage d'isolats résistants est resté élevé (17,7% (IC95%: 11,1-27,1%) au Burkina Faso et 19,1% (IC95%: 13,5-26,3%) au Mali) chez les enfants ayant reçu l'azithromycine un an après arrêter l'intervention. Une augmentation de la résistance à l'azithromycine a également été observée chez les enfants ayant reçu un placebo, mais elle était moins marquée.L’ajout d'azithromycine à la combinaison antipaludique utilisée pour la chimioprévention du paludisme saisonnier était associé à une augmentation de la résistance du pneumocoque à l'azithromycine et à l'érythromycine, qui persistait un an après la dernière administration d'azithromycine.

Malaria, a vector borne disease caused by Plasmodium spp., remains a major global cause of morbidity and mortality. Optimization of the disease control strategies requires a thorough understanding of the fundamental processes underlying parasite transmission. Although the number of transmissible stages of Plasmodium (gametocyte) in human blood is frequently used as an indicator of human-to-mosquito transmission potential, this relationship is not always clear. Important efforts have been made to develop molecular tools to fine-tune gametocyte densities estimation and therefore improve the prediction of mosquito infection rates, but a significant level of uncertainty around this estimate remains. Here we show with both human and avian malaria system that the within-vertebrate host distribution of gametocytes could explain much of this uncertainty. By comparing gametocyte densities in bloodstream between different body parts, we found a difference by nearly 50% in humans and by more than 15% in birds. An estimation of gametocyte density from only one blood sample, as is usually the case, could therefore drastically over- or underestimated the infectivity of gametocyte carriers. This might have important consequences on the epidemiology of the disease since we show, using the avian malaria system, that this variation influences the transmission of the parasite to the mosquito vector. In the light of our results, we argue that it is essential to consider the heterogeneous distribution of gametocyte to improve human diagnosis, identify infectious reservoirs and to test new malaria control strategies.

Background Medicinal plant research may contribute to develop new pharmacological control tools for vector borne diseases, such as malaria. Methods The effects of methanol extracts (ME) obtained from seed kernel of ripe and unripe Azadirachta indica fruits were studied on erythrocytic proliferation of the rodent malaria parasite Plasmodium berghei strain ANKA and on mice pro-inflammatory response, as evaluated by measuring the matrix-metalloproteinase-9 (MMP-9) and tumour necrosis factor (TNF) plasma levels, in two mouse strains (C57BL/6 and BALB/c) which are considered as prototypical of Th1 and Th2 immune response, respectively. Results ME obtained from seed kernel of unripe Azadirachta indica fruits decreased by about 30% the proportion of erythrocytes infected with the malaria parasite in C57BL/6 mice in the 4 days suppressive test. In this treatment group, MMP-9 and TNF levels were notably higher than those measured in the same mouse strain treated with the anti-malarial drug artesunate, Azadirachta indica kernel extracts from ripe fruits or solvent. In BALB/c mice, treatment with kernel extracts did not influence parasitaemia. MMP-9 and TNF levels measured in this mouse strain were notably lower than those recorded in C57BL/6 mice and did not vary among treatment groups. Conclusions The effects of the ME on the parasite-host interactions appeared to be mouse strain-dependent, but also related to the ripening stage of the neem fruits, as only the unripe fruit seed kernel extracts displayed appreciable bioactivity. Electronic supplementary material The online version of this article (10.1186/s12936-019-2671-8) contains supplementary material, which is available to authorized users.

Objective Cassia alata L. (Fabaceae), one of the three plants contained in Saye, a polyherbal antimalarial remedy was assessed for its antimalarial potential and safety in mice. Methodology Organic extracts were prepared from the leaves and tested on the D 10 chloroquine-sensitive strain of Plasmodium falciparum using the parasite lactate dehydrogenase assay. The 4 days suppressive test using Plasmodium berghei in mice was used to evaluate the in vivo antiplasmodial activity of the extracts. Animals were treated by oral route, once a day with 50, 100, 250 and 400 mg kg -1 b.wt., of the extracts. The acute toxicity of the extracts was assessed in mice according to Thompson and Weil method. The lethal effects of the extracts on animal's body weight, tissues, biochemical and haematological parameters were determined at 823.5, 1235.5, 1853 and 2779.5 mg kg -1 b.wt., respectively. Results The dichloromethane/methane (1:1, v/v) extract of Cassia alata was the most active against Plasmodium falciparum. The mean percent suppression of parasitemia in mice was equal to 22.5, 41.8 and 45.2% at 50, 250 and 400 mg kg -1 b.wt., respectively. No death and no clinically significant changes were recorded in mice. The maximum non-lethal dose was more than 16875 mg kg -1 in animals. No significant changes were observed in body weight, tissues morphology, biochemical and hematological parameters at doses above or equal to 2779.5 mg kg -1 b.wt. Conclusion The dichloromethane/methanol leaf extract of Cassia alata had a good to moderate in vitro and in vivo antiplasmodial activity and was found to have low toxicity at high doses in tested animals.

Anopheles mosquitoes have transmitted Plasmodium parasites for millions of years, yet it remains unclear whether they suffer fitness costs to infection. Here we report that the fecundity of virgin and mated females of two important vectors—Anopheles gambiae and Anopheles stephensi—is not affected by infection with Plasmodium falciparum, demonstrating that these human malaria parasites do not inflict this reproductive cost on their natural mosquito hosts. Additionally, parasite development is not impacted by mating status. However, in field studies using different P. falciparum isolates in Anopheles coluzzii, we find that Mating-Induced Stimulator of Oogenesis (MISO), a female reproductive gene strongly induced after mating by the sexual transfer of the steroid hormone 20-hydroxyecdysone (20E), protects females from incurring fecundity costs to infection. MISO-silenced females produce fewer eggs as they become increasingly infected with P. falciparum, while parasite development is not impacted by this gene silencing. Interestingly, previous work had shown that sexual transfer of 20E has specifically evolved in Cellia species of the Anopheles genus, driving the co-adaptation of MISO. Our data therefore suggest that evolution of male-female sexual interactions may have promoted Anopheles tolerance to P. falciparum infection in the Cellia subgenus, which comprises the most important malaria vectors., Author summary Plasmodium falciparum, the deadliest form of human malaria, is transmitted when female Anopheles mosquitoes bite people and take a blood meal in order to develop eggs. To date, it is still poorly understood whether Anopheles mosquitoes that get infected with P. falciparum suffer fitness costs. Here, we find that the number of eggs produced by Anopheles gambiae and Anopheles stephensi females is not affected by P. falciparum infection, and that the mating status of the mosquitoes does not impact the parasite. However, in field experiments infecting a related species, Anopheles coluzzii, with P. falciparum using blood from donors in Burkina Faso, we find that interfering with the expression of a gene normally triggered by the sexual transfer of the steroid hormone 20-hydroxyecdysone induces increasing costs to egg development as females become more infected with P. falciparum, with no impacts on the parasite. The results of our study suggest that pathways triggered by mating may help Anopheles prevent reproductive costs associated with P. falciparum infection, providing new insights into evolutionary strategies adopted by anophelines in the face of a longstanding association with Plasmodium parasites.

[Background] Infection by the human malaria parasite leads to important changes in mosquito phenotypic traits related to vector competence. However, we still lack a clear understanding of the underlying mechanisms and, in particular, of the epigenetic basis for these changes. We have examined genome-wide distribution maps of H3K27ac, H3K9ac, H3K9me3 and H3K4me3 by ChIP-seq and the transcriptome by RNA-seq, of midguts from Anopheles gambiae mosquitoes blood-fed uninfected and infected with natural isolates of the human malaria parasite Plasmodium falciparum in Burkina Faso., [Results] We report 15,916 regions containing differential histone modification enrichment between infected and uninfected, of which 8339 locate at promoters and/or intersect with genes. The functional annotation of these regions allowed us to identify infection-responsive genes showing differential enrichment in various histone modifications, such as CLIP proteases, antimicrobial peptides-encoding genes, and genes related to melanization responses and the complement system. Further, the motif analysis of regions differentially enriched in various histone modifications predicts binding sites that might be involved in the cis-regulation of these regions, such as Deaf1, Pangolin and Dorsal transcription factors (TFs). Some of these TFs are known to regulate immunity gene expression in Drosophila and are involved in the Notch and JAK/STAT signaling pathways., [Conclusions] The analysis of malaria infection-induced chromatin changes in mosquitoes is important not only to identify regulatory elements and genes underlying mosquito responses to P. falciparum infection, but also for possible applications to the genetic manipulation of mosquitoes and to other mosquito-borne systems., This work was supported by the Spanish Ministry of Economy and Competitiveness Grant BFU2015-65000-R to EG-D; Severo Ochoa Fellowship BES-2016-076276 to JLR; Spanish Ministry of Economy and Competitiveness Ramon y Cajal Grant to EG-D; French ANR grant 11-PDOC-006-01 to TL and PATH Malaria Vaccine Initiative to TL, JBO and RSY, and by U.S. Public Health Service Award R01GM035463 from the National Institutes of Health to VGC.

Infection by the human malaria parasite leads to important changes in mosquito phenotypic traits related to vector competence. However, we still lack a clear understanding of the underlying mechanisms and in particular, of the epigenetic basis for these changes. We have examined genome-wide distribution maps of H3K27ac, H3K9ac, H3K9me3 and H3K4me3 by ChIP-seq and the transcriptome by RNA-seq, of midguts from Anopheles gambiae mosquitoes infected with natural isolates of the human malaria parasite Plasmodium falciparum in Burkina Faso. We report 15,916 regions containing differential histone modification enrichment, of which 8,339 locate at promoters and/or intersect with genes. The functional annotation of these regions allowed us to identify infection responsive genes showing differential enrichment in various histone modifications, such as CLIP pro-teases, anti-microbial peptides encoding genes, and genes related to melanization responses and the complement system. Further, the motif analysis of regions differentially enriched in various histone modifications predicts binding sites that might be involved in the cis-regulation of these regions such as Deaf1, Pangolin and Dorsal transcription factors (TFs). Some of these TFs are known to regulate immunity gene expression in Drosophila and are involved in the Notch and JAK/STAT signaling pathways. The analysis of malaria infection-induced chromatin changes in mosquitoes is important not only to identify regulatory elements and genes underlying mosquito responses to a P. falciparum infection but also for possible applications to the genetic manipulation of mosquitoes and to other mosquito-borne systems.

During the 20th century, health inequalities among countries have increased. Several factors explain this pattern, such as immunization and massive antibiotherapy, but nutrition, housing and hygiene are key parameters for health improvement. This heterogeneity among countries is well illustrated by malaria, although disappeared from many high-income countries, is still endemic and prevalent in low- and middle-income countries. We question these differences and detail the recommendations proposed by the World Health Organization to tackle malaria. We investigate the optimal combination of actions to deploy in resource-limited countries and the best spatio-temporal window to target. We propose a new framework for health program management based on evolutionary biology approaches to tailor global programs, to improve their local efficiency and avoid resistance. Thus, we explore all components of the ecological niche of the parasite (human, vector and environment) and consider the magnitude of actions to deploy to reach its local.

The evaluation of transmission reducing interventions (TRI) to control malaria widely uses membrane feeding assays. In such assays, the intensity of Plasmodium infection in the vector might affect the measured efficacy of the candidates to block transmission. Gametocyte density in the host blood is a determinant of the infection success in the mosquito, however, uncertain estimates of parasite densities and intrinsic characteristics of the infected blood can induce variability. To reduce this variation, a feasible method is to dilute infectious blood samples. We describe the effect of diluting samples of Plasmodium -containing blood samples to allow accurate relative measures of gametocyte densities and their impact on mosquito infectivity and TRI efficacy. Natural Plasmodium falciparum samples were diluted to generate a wide range of parasite densities, and fed to Anopheles coluzzii mosquitoes. This was compared with parallel dilutions conducted on Plasmodium berghei infections. We examined how blood dilution influences the observed blocking activity of anti-Pbs28 monoclonal antibody using the P. berghei/Anopheles stephensi system. In the natural species combination P. falciparum/An. coluzzii , blood dilution using heat-inactivated, infected blood as diluents, revealed positive near linear relationships, between gametocyte densities and oocyst loads in the range tested. A similar relationship was observed in the P. berghei/An. stephensi system when using a similar dilution method. In contrast, diluting infected mice blood with fresh uninfected blood dramatically increases the infectiousness. This suggests that highly infected mice blood contains inhibitory factors or reduced blood moieties, which impede infection and may in turn, lead to misinterpretation when comparing individual TRI evaluation assays. In the lab system, the transmission blocking activity of an antibody specific for Pbs28 was confirmed to be density-dependent. This highlights the need to carefully interpret evaluations of TRI candidates, regarding gametocyte densities in the P. berghei/An. stephensi system.

Background: The bacterial microbiota which colonize the mosquito midgut play an important role in vector-parasite interactions and consequently can modulate the level of malaria transmission. Their characterization may contribute to new control strategies of malaria transmission. However, these bacteria may also be eliminated in areas of high antibiotics usage. In this study, we identified paratransgenesis bacteria candidate in the gut of adults female Anopheles in Burkina Faso. Methods: The gut of 73 semi-field mosquitoes and 28 laboratory-reared mosquitoes from two villages in Burkina Faso were analyzed by conventional in vitro culture techniques to isolate and identify bacteria of the microbiota. The gene 16S sequencing was used to confirm the presence of bacteria of paratransgenesis interest. Due to the effect of antibiotics on bacteria, we evaluated in vitro their susceptibility to antibiotics generally used for infectious diseases treatment. Results: In total, eleven genera of bacteria were identified: Pantoea, Sphingomonas, Escherichia, Micrococcus, Staphylococcus, Klebsiella, Serratia, Acinetobacter, Pseudomonas, Citrobacter, Asaia. Among these bacteria isolated, Asaia. sp and Pantoea. sp have already been reported as candidates for paratransgenesis. In addition, we observed pathogenic bacteria such as Escherichia coli, Klebsiella pneumonia and Pseudomonas luteola. Investigation of the correlation between the bacterial microbiota and malaria infection status showed that mosquitoes engorged with blood containing Plasmodium falciparum contained a higher bacterial load than non-blood fed mosquitoes. The antibiotic susceptibility test showed that Asaia, Pantoea and Serratia, previously proposed as paratransgenesis candidates, were susceptible to different antibiotics tested in contrast to Escherichia coli, which were resistant. Discussion: Midgut analysis shows that the composition of the bacterial microbiota in wild field mosquitoes exhibits a large variability in contrast to laboratory-reared mosquitoes. The presence of genera already proposed as paratransgenesis candidates in previous studies among bacteria isolated in our study, suggested the possible implementation of this control strategy in Burkina Faso. Nevertheless, our data indicates that an in vivo verification of the stability of these bacteria is needed, as this strategy may be impaired by mass drug administration programs and antibiotic misuse.

P. falciparum phenotypic plasticity is linked to the variant expression of clonal multigene families such as the var genes. We have examined changes in transcription and histone modifications that occur during sporogonic development of P. falciparum in the mosquito host. All var genes are silenced or transcribed at low levels in blood stages (gametocyte/ring) of the parasite in the human host. After infection of mosquitoes, a single var gene is selected for expression in the oocyst, and transcription of this gene increases dramatically in the sporozoite. The same PF3D7_1255200 var gene was activated in 4 different experimental infections. Transcription of this var gene during parasite development in the mosquito correlates with the presence of low levels of H3K9me3 at the binding site for the PF3D7_1466400 AP2 transcription factor. This chromatin state in the sporozoite also correlates with the expression of an antisense long non-coding RNA (lncRNA) that has previously been shown to promote var gene transcription during the intraerythrocytic cycle in vitro. Expression of both the sense proteincoding transcript and the antisense lncRNA increase dramatically in sporozoites. The findings suggest a complex process for the activation of a single particular var gene that involves AP2 transcription factors and lncRNAs

phytochemical assay was conducted to establish the chemical profile of “Saye”, a mixture of leaf of Cassia alata, root of Cochlospermum planchonii and whole plant of Phyllantus amarus , used as antimarial remedy. Water and organic extracts were prepared. Characterization of phytoconstituents using specific chemical reagents was performed in tubes, by thin layer chromatography and by high performance liquid chromatography. Steroids and/or triterpenes, cathechic tannins were identified in the decocted and the macerated water extracts of “Saye”. An anthraquinone with a retention time Rt corresponding to 3.34 min was identified by the HPLC analysis. © 2015 International Formulae Group. All rights reserved. Keywords : Chemical profile, anthraquinones, steroids, triterpenes, tannins.

Ethnopharmacological relevance Saye, a combination remedy prepared from Cochlospermum planchonii Hook.f. (Cochlospermaceae), Cassia alata L. (Fabaceae) and Phyllanthus amarus Schumach. et Thonn. (Euphorbiaceae), N’Dribala, a Cochlospermum planchonii root decoction, and a fruit preparation of Azadirachta indica A. Juss. (Meliaceae) are plant remedies of the folk medicine in Burkina Faso and are commonly used by traditional healers for the treatment of malaria. Aim of the study This study aimed at validating the antiplasmodial activity of the preparations and at estimating their potential for prophylaxis, using the murine malaria system Plasmodium berghei/Anopheles stephensi. Materials and methods Aqueous extracts were orally administered to mice (6 animals per treatment group) at a daily dose of 200 mg/kg body weight for nine days, applying protocols that mimic as much as possible traditional recipes and treatment schemes. Results Saye, N’Dribala and Azadirachta indica preparations revealed prophylactic activity, reducing parasitaemia in treated mice, with respect to controls, by 52.0% (CI95 46.1–57.9), 45.5% (CI95 44.5–46.5) and 45.0% (CI95 41.1–48.9), respectively. No evidence of transmission blocking effects was detected with any of the tested remedies. Conclusions This study confirms, in the murine malaria system, the antiplasmodial properties of the examined remedies on the Plasmodium stages developing in the vertebrate host, thus encouraging studies aiming at identifying the active fractions and compounds responsible for the described activity and to develop standardized prophylactic remedies.

The ecological context in which mosquitoes and malaria parasites interact has received little attention, compared to the genetic and molecular aspects of malaria transmission. Plant nectar and fruits are important for the nutritional ecology of malaria vectors, but how the natural diversity of plant-derived sugar sources affects mosquito competence for malaria parasites is unclear. To test this, we infected Anopheles coluzzi, an important African malaria vector, with sympatric field isolates of Plasmodium falciparum, using direct membrane feeding assays. Through a series of experiments, we then examined the effects of sugar meals from Thevetia neriifolia and Barleria lupilina cuttings that included flowers, and fruit from Lannea microcarpa and Mangifera indica on parasite and mosquito traits that are key for determining the intensity of malaria transmission. We found that the source of plant sugar meal differentially affected infection prevalence and intensity, the development duration of the parasites, as well as the survival and fecundity of the vector. These effects are likely the result of complex interactions between toxic secondary metabolites and the nutritional quality of the plant sugar source, as well as of host resource availability and parasite growth. Using an epidemiological model, we show that plant sugar source can be a significant driver of malaria transmission dynamics, with some plant species exhibiting either transmission-reducing or -enhancing activities., Author Summary The Anopheles coluzzii mosquito is an effective vector of human Plasmodium falciparum malaria. Besides feeding on blood, females readily feed on natural sources of plant sugars; but how plant diversity affects their ability to transmit malaria parasites is currently unknown. Here we show that mosquito feeding on peridomestic plant- and fruit-derived sugar sources can affect its interaction with P. falciparum, and hence malaria transmission. The planting of anti-Plasmodium plant sugar sources may represent a promising alternative strategy to contribute to the control of malaria.

Azadirachta indica, known as neem tree and traditionally called “nature's drug store” makes part of several African pharmacopeias and is widely used for the preparation of homemade remedies and commercial preparations against various illnesses, including malaria. Employing a bio-guided fractionation approach, molecules obtained from A. indica ripe and green fruit kernels were tested for activity against early sporogonic stages of Plasmodium berghei, the parasite stages that develop in the mosquito mid gut after an infective blood meal. The limonoid deacetylnimbin (3) was identified as one the most active compounds of the extract, with a considerably higher activity compared to that of the close analogue nimbin (2). Pure deacetylnimbin (3) appeared to interfere with transmissible Plasmodium stages at a similar potency as azadirachtin A. Considering its higher thermal and chemical stability, deacetylnimbin could represent a suitable alternative to azadirachtin A for the preparation of transmission blocking antimalarials.

Malaria reduction is most efficiently achieved by vector control whereby human populations at high risk of contracting and transmitting the disease are protected from mosquito bites. Here, we identify the presence of antibiotics in the blood of malaria-infected people as a new risk of increasing disease transmission. We show that antibiotics in ingested blood enhance the susceptibility of Anopheles gambiae mosquitoes to malaria infection by disturbing their gut microbiota. This effect is confirmed in a semi-natural setting by feeding mosquitoes with blood of children naturally infected with Plasmodium falciparum. Antibiotic exposure additionally increases mosquito survival and fecundity, which are known to augment vectorial capacity. These findings suggest that malaria transmission may be exacerbated in areas of high antibiotic usage, and that regions targeted by mass drug administration programs against communicable diseases may necessitate increased vector control., The gut microbiota of malaria-transmitting mosquitoes contributes to the insects’ resistance to the parasite. Here, Gendrin et al. show that antibiotics in ingested human blood alter the mosquito gut microbiota and increase the insect’s survival, fecundity and susceptibility to the parasites.

Background: Targeting the stages of the malaria parasites responsible for transmission from the human host to the mosquito vector is a key pharmacological strategy for malaria control. Research efforts to identify compounds that are active against these stages have significantly increased in recent years. However, at present, only two drugs are available, namely primaquine and artesunate, which reportedly act on late stage gametocytes. Methods: In this study, we assessed the antiplasmodial effects of 5 extracts obtained from the neem tree Azadirachta indica and Guiera senegalensis against the early vector stages of Plasmodium falciparum, using field isolates. In an ex vivo assay gametocytaemic blood was supplemented with the plant extracts and offered to Anopheles coluzzii females by membrane feeding. Transmission blocking activity was evaluated by assessing oocyst prevalence and density on the mosquito midguts. Results: Initial screening of the 5 plant extracts at 250 ppm revealed transmission blocking activity in two neem preparations. Up to a concentration of 70 ppm the commercial extract NeemAzal (R) completely blocked transmission and at 60 ppm mosquitoes of 4 out of 5 replicate groups remained uninfected. Mosquitoes fed on the ethyl acetate phase of neem leaves at 250 ppm showed a reduction in oocyst prevalence of 59.0% (CI95 12.0 -79.0; p < 10(-4)) and in oocyst density of 90.5% (CI95 86.0 -93.5; p < 10(-4)), while the ethanol extract from the same plant part did not exhibit any activity. No evidence of transmission blocking activity was found using G. senegalensis ethyl acetate extract from stem galls. Conclusions: The results of this study highlight the potential of antimalarial plants for the discovery of novel transmission blocking molecules, and open up the potential of developing standardized transmission blocking herbal formulations as malaria control tools to complement currently used antimalarial drugs and combination treatments.

Widespread and common across much of the drier areas of western Africa, the woody shrub Guiera senegalensis (Combretaceae) is the sole member of its genus. Similarly widespread is Vuilletia houardi, a thrips species that induces galls on this shrub, and is recorded from Mali, Senegal, Gambia and northern Nigeria (Pitkin & Mound 1973). Moreover, large numbers of galls, together with their included thrips, have now been studied from Burkina Faso. Some galls (Figs 1, 2) are invaded by Senegathrips coutini, a species whose biology is not known but that is possibly a predator. Moreover, Liothrips africana also sometimes breeds within these galls, but is possibly using these only as a convenient shelter. A re-description and line-drawings of V. houardi was provided by zur Strassen (1958), but no modern diagnosis of this genus, nor of Senegathrips, is available, the objective here being to provide formal diagnoses for these two monotypic genera.

Background The wide use of gametocytocidal artemisinin-based combination therapy (ACT) lead to a reduction of Plasmodium falciparum transmission in several African endemic settings. An increased impact on malaria burden may be achieved through the development of improved transmission-blocking formulations, including molecules complementing the gametocytocidal effects of artemisinin derivatives and/or acting on Plasmodium stages developing in the vector. Azadirachtin, a limonoid (tetranortriterpenoid) abundant in neem (Azadirachta indica, Meliaceae) seeds, is a promising candidate, inhibiting Plasmodium exflagellation in vitro at low concentrations. This work aimed at assessing the transmission-blocking potential of NeemAzal®, an azadirachtin-enriched extract of neem seeds, using the rodent malaria in vivo model Plasmodium berghei/Anopheles stephensi. Methods Anopheles stephensi females were offered a blood-meal on P. berghei infected, gametocytaemic BALB/c mice, treated intraperitoneally with NeemAzal, one hour before feeding. The transmission-blocking activity of the product was evaluated by assessing oocyst prevalence, oocyst density and capacity to infect healthy mice. To characterize the anti-plasmodial effects of NeemAzal® on early midgut stages, i.e. zygotes and ookinetes, Giemsa-stained mosquito midgut smears were examined. Results NeemAzal® completely blocked P. berghei development in the vector, at an azadirachtin dose of 50 mg/kg mouse body weight. The totally 138 examined, treated mosquitoes (three experimental replications) did not reveal any oocyst and none of the healthy mice exposed to their bites developed parasitaemia. The examination of midgut content smears revealed a reduced number of zygotes and post-zygotic forms and the absence of mature ookinetes in treated mosquitoes. Post-zygotic forms showed several morphological alterations, compatible with the hypothesis of an azadirachtin interference with the functionality of the microtubule organizing centres and with the assembly of cytoskeletal microtubules, which are both fundamental processes in Plasmodium gametogenesis and ookinete formation. Conclusions This work demonstrated in vivo transmission blocking activity of an azadirachtin-enriched neem seed extract at an azadirachtin dose compatible with 'druggability' requisites. These results and evidence of anti-plasmodial activity of neem products accumulated over the last years encourage to convey neem compounds into the drug discovery & development pipeline and to evaluate their potential for the design of novel or improved transmission-blocking remedies.