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.

Multiple insecticide resistance and first evidence of V410L kdr mutation in Aedes (Stegomyia) aegypti (Linnaeus) from Burkina Faso.

The response to recent dengue outbreaks in Burkina Faso was insecticide-based, despite poor knowledge of the vector population's susceptibility to the insecticides used. Here, we report on the susceptibility to the main insecticide classes and identify important underlying mechanisms in Aedes aegypti populations in Ouagadougou and Banfora, in 2019 and 2020. Wild Ae. aegypti were tested as adults in WHO bioassays and then screened in real time melting curve qPCR analyses to genotype the F1534C, V1016I, and V410L Aedes kdr mutations. Ae. aegypti showed moderate resistance to 0.1% bendiocarb (80-95% survival post-exposure), 0.8% Malathion (60-100%), 0.21% pirimiphos-methyl (75% - 97%), and high resistance to 0.03% deltamethrin (20-70%). PBO pre-exposure partially restored pyrethroid susceptibility. Genotyping detected high frequency of 1534C allele (0.92) and moderate 1016I (0.1-0.32). The V410L mutation was detected in Burkina Faso for the first time (frequency 0.1-0.36). Mosquitoes surviving 4 h exposure to 0.03% deltamethrin had significantly higher frequencies of the F1534C mutation than dead mosquitoes (0.70 vs. 0.96, p < 0.0001) and mosquitoes surviving 2 - 4 h exposure had a significantly reduced life span. Ae. aegypti from Burkina Faso are resistant to multiple insecticide classes with multiple mechanisms involved, demonstrating the essential role of insecticide resistance monitoring within national dengue control programmes.

Comprehensive Ecological and Geographic Characterization of Eukaryotic and Prokaryotic Microbiomes in African Anopheles.

Exposure of mosquitoes to numerous eukaryotic and prokaryotic microbes in their associated microbiomes has probably helped drive the evolution of the innate immune system. To our knowledge, a metagenomic catalog of the eukaryotic microbiome has not been reported from any insect. Here we employ a novel approach to preferentially deplete host 18S ribosomal RNA gene amplicons to reveal the composition of the eukaryotic microbial communities of Anopheles larvae sampled in Kenya, Burkina Faso and Republic of Guinea (Conakry). We identified 453 eukaryotic operational taxonomic units (OTUs) associated with Anopheles larvae in nature, but an average of 45% of the 18S rRNA sequences clustered into OTUs that lacked a taxonomic assignment in the Silva database. Thus, the Anopheles microbiome contains a striking proportion of novel eukaryotic taxa. Using sequence similarity matching and de novo phylogenetic placement, the fraction of unassigned sequences was reduced to an average of 4%, and many unclassified OTUs were assigned as relatives of known taxa. A novel taxon of the genus Ophryocystis in the phylum Apicomplexa (which also includes Plasmodium) is widespread in Anopheles larvae from East and West Africa. Notably, Ophryocystis is present at fluctuating abundance among larval breeding sites, consistent with the expected pattern of an epidemic pathogen. Species richness of the eukaryotic microbiome was not significantly different across sites from East to West Africa, while species richness of the prokaryotic microbiome was significantly lower in West Africa. Laboratory colonies of Anopheles coluzzii harbor 26 eukaryotic OTUs, of which 38% (n = 10) are shared with wild populations, while 16 OTUs are unique to the laboratory colonies. Genetically distinct An. coluzzii colonies co-housed in the same facility maintain different prokaryotic microbiome profiles, suggesting a persistent host genetic influence on microbiome composition. These results provide a foundation to understand the role of the Anopheles eukaryotic microbiome in vector immunity and pathogen transmission. We hypothesize that prevalent apicomplexans such as Ophryocystis associated with Anopheles could induce interference or competition against Plasmodium within the vector. This and other members of the eukaryotic microbiome may offer candidates for new vector control tools.

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.

Comprehensive Genomic Discovery of Non-Coding Transcriptional Enhancers in the African Malaria Vector Anopheles coluzzii.

Almost all regulation of gene expression in eukaryotic genomes is mediated by the action of distant non-coding transcriptional enhancers upon proximal gene promoters. Enhancer locations cannot be accurately predicted bioinformatically because of the absence of a defined sequence code, and thus functional assays are required for their direct detection. Here we used a massively parallel reporter assay, Self-Transcribing Active Regulatory Region sequencing (STARR-seq), to generate the first comprehensive genome-wide map of enhancers in Anopheles coluzzii, a major African malaria vector in the Gambiae species complex. The screen was carried out by transfecting reporter libraries created from the genomic DNA of 60 wild A. coluzzii from Burkina Faso into A. coluzzii 4a3A cells, in order to functionally query enhancer activity of the natural population within the homologous cellular context. We report a catalog of 3,288 active genomic enhancers that were significant across three biological replicates, 74% of them located in intergenic and intronic regions. The STARR-seq enhancer screen is chromatin-free and thus detects inherent activity of a comprehensive catalog of enhancers that may be restricted in vivo to specific cell types or developmental stages. Testing of a validation panel of enhancer candidates using manual luciferase assays confirmed enhancer function in 26 of 28 (93%) of the candidates over a wide dynamic range of activity from two to at least 16-fold activity above baseline. The enhancers occupy only 0.7% of the genome, and display distinct composition features. The enhancer compartment is significantly enriched for 15 transcription factor binding site signatures, and displays divergence for specific dinucleotide repeats, as compared to matched non-enhancer genomic controls. The genome-wide catalog of A. coluzzii enhancers is publicly available in a simple searchable graphic format. This enhancer catalogue will be valuable in linking genetic and phenotypic variation, in identifying regulatory elements that could be employed in vector manipulation, and in better targeting of chromosome editing to minimize extraneous regulation influences on the introduced sequences. Importance: Understanding the role of the non-coding regulatory genome in complex disease phenotypes is essential, but even in well-characterized model organisms, identification of regulatory regions within the vast non-coding genome remains a challenge. We used a large-scale assay to generate a genome wide map of transcriptional enhancers. Such a catalogue for the important malaria vector, Anopheles coluzzii, will be an important research tool as the role of non-coding regulatory variation in differential susceptibility to malaria infection is explored and as a public resource for research on this important insect vector of disease.

Evaluation of mosquito electrocuting traps as a safe alternative to the human landing catch for measuring human exposure to malaria vectors in Burkina Faso.

BACKGROUND: Measuring human exposure to mosquito bites is a crucial component of vector-borne disease surveillance. For malaria vectors, the human landing catch (HLC) remains the gold standard for direct estimation of exposure. This method, however, is controversial since participants risk exposure to potentially infected mosquito bites. Recently an exposure-free mosquito electrocuting trap (MET) was developed to provide a safer alternative to the HLC. Early prototypes of the MET performed well in Tanzania but have yet to be tested in West Africa, where malaria vector species composition, ecology and behaviour are different. The performance of the MET relative to HLC for characterizing mosquito vector population dynamics and biting behaviour in Burkina Faso was evaluated. METHODS: A longitudinal study was initiated within 12 villages in Burkina Faso in October 2016. Host-seeking mosquitoes were sampled monthly using HLC and MET collections over 14 months. Collections were made at 4 households on each night, with METs deployed inside and outside at 2 houses, and HLC inside and outside at another two. Malaria vector abundance, species composition, sporozoite rate and location of biting (indoor versus outdoor) were recorded. RESULTS: In total, 41,800 mosquitoes were collected over 324 sampling nights, with the major malaria vector being Anopheles gambiae sensu lato (s.l.) complex. Overall the MET caught fewer An. gambiae s.l. than the HLC (mean predicted number of 0.78 versus 1.82 indoors, and 1.05 versus 2.04 outdoors). However, MET collections gave a consistent representation of seasonal dynamics in vector populations, species composition, biting behaviour (location and time) and malaria infection rates relative to HLC. As the relative performance of the MET was somewhat higher in outdoor versus indoor settings, this trapping method slightly underestimated the proportion of bites preventable by LLINs compared to the HLC (MET = 82.08%; HLC = 87.19%). CONCLUSIONS: The MET collected proportionately fewer mosquitoes than the HLC. However, estimates of An. gambiae s.l. density in METs were highly correlated with HLC. Thus, although less sensitive, the MET is a safer alternative than the HLC. Its use is recommended particularly for sampling vectors in outdoor environments where it is most sensitive.

Influence of genetic polymorphism on transcriptional enhancer activity in the malaria vector Anopheles coluzzii.

Enhancers are cis-regulatory elements that control most of the developmental and spatial gene expression in eukaryotes. Genetic variation of enhancer sequences is known to influence phenotypes, but the effect of enhancer variation upon enhancer functional activity and downstream phenotypes has barely been examined in any species. In the African malaria vector, Anopheles coluzzii, we identified candidate enhancers in the proximity of genes relevant for immunity, insecticide resistance, and development. The candidate enhancers were functionally validated using luciferase reporter assays, and their activity was found to be essentially independent of their physical orientation, a typical property of enhancers. All of the enhancers segregated genetically polymorphic alleles, which displayed significantly different levels of functional activity. Deletion mutagenesis and functional testing revealed a fine structure of positive and negative regulatory elements that modulate activity of the enhancer core. Enhancer polymorphisms segregate in wild A. coluzzii populations in West Africa. Thus, enhancer variants that modify target gene expression leading to likely phenotypic consequences are frequent in nature. These results demonstrate the existence of naturally polymorphic A. coluzzii enhancers, which may help explain important differences between individuals or populations for malaria transmission efficiency and vector adaptation to the environment.

Anopheline species composition and the 1014F-genotype in different ecological settings of Burkina Faso in relation to malaria transmission.

BACKGROUND: A three-year longitudinal study was conducted in four sentinel sites from different ecological settings in Burkina Faso, between 2008 and 2010 to identify longitudinal changes in insecticide resistance within Anopheles gambiae complex species based on larval collection. During this study, adult mosquitoes were also collected indoor and outdoor using several methods of collection. The present study reports the diversity of malaria vectors and the 1014F-genotype from this adult collection and investigates the association between this 1014F-genotype and sporozoite rate. METHODS: Adult mosquitoes were collected from July to August (corresponding to the start of rainy season) and October to November (corresponding to the end of rainy season) over 3 years (2008-2010) at four sites across the country, using pyrethrum spray catches (PSC), exit traps and pit shelters. Anopheles gambiae complex mosquitoes were identified to species and genotyped for the L1014F kdr mutation by PCR using genomic DNA. The circumsporozoite antigen of Plasmodium falciparum was detected in mosquitoes using sandwich ELISA. RESULTS: Overall 9212 anopheline mosquitoes were collected during the study period. Of those, 6767 mosquitoes were identified as Anopheles gambiae sensu lato (s.l.). Anopheles arabiensis, Anopheles coluzzii, Anopheles gambiae and or Anopheles funestus were incriminated as vectors of P. falciparum in the study area with an average sporozoite rate of 5%, (95% CI 4.14-5.99%). The kdr1014F-genotype frequencies were 11.44% (95% CI 2.5-39.85%), 19.2% (95% CI 4.53-53.73%) and 89.9 (95% CI 63.14-97.45%), respectively for An. arabiensis, An. coluzzii and An. gambiae. The proportion of the 1014F-genotype varied between sporozoite-infected and uninfected An. gambiae s.l. group. There was no significant difference in the 1014F-genotype frequency between infected and uninfected mosquitoes. CONCLUSION: The current study shows the diversity of malaria vectors and significant interaction between species composition and kdr1014F-genotype in An. gambiae complex mosquitoes from Burkina Faso. In this study, no associations were found between the 1014F-genotype and P. falciparum infection in the major malaria vector An. gambiae s.l.

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.

Equivalent susceptibility of Anopheles gambiae M and S molecular forms and Anopheles arabiensis to Plasmodium falciparum infection in Burkina Faso.

BACKGROUND: The Anopheles gambiae sensu lato (s.l.) species complex in Burkina Faso consists of Anopheles arabiensis, and molecular forms M and S of Anopheles gambiae sensu stricto (s.s.). Previous studies comparing the M and S forms for level of infection with Plasmodium falciparum have yielded conflicting results. METHODS: Mosquito larvae were sampled from natural pools, reared to adulthood under controlled conditions, and challenged with natural P. falciparum by experimental feeding with blood from gametocyte carriers. Oocyst infection prevalence and intensity was determined one week after infection. DNA from carcasses was genotyped to identify species and molecular form. RESULTS: In total, 7,400 adult mosquitoes grown from wild-caught larvae were challenged with gametocytes in 29 experimental infections spanning four transmission seasons. The overall infection prevalence averaged 40.7% for A. gambiae M form, 41.4% for A. gambiae S form, and 40.1% for A. arabiensis. There was no significant difference in infection prevalence or intensity between the three population groups. Notably, infection experiments in which the population groups were challenged in parallel on the same infective blood displayed less infection difference between population groups, while infections with less balanced composition of population groups had lower statistical power and displayed apparent differences that fluctuated more often from the null average. CONCLUSION: The study clearly establishes that, at the study site in Burkina Faso, there is no difference in genetic susceptibility to P. falciparum infection between three sympatric population groups of the A. gambiae s.l. complex. Feeding the mosquito groups on the same infective blood meal greatly increases statistical power. Conversely, comparison of the different mosquito groups between, rather than within, infections yields larger apparent difference between mosquito groups, resulting from lower statistical power and greater noise, and could lead to false-positive results. In making infection comparisons between population groups, it is more accurate to compare the different groups after feeding simultaneously upon the same infective blood.