Mosquito control by abatement programmes in the United States: perspectives and lessons for countries in sub-Saharan Africa.

Africa and the United States are both large, heterogeneous geographies with a diverse range of ecologies, climates and mosquito species diversity which contribute to disease transmission and nuisance biting. In the United States, mosquito control is nationally, and regionally coordinated and in so much as the Centers for Disease Control (CDC) provides guidance, the Environmental Protection Agency (EPA) provides pesticide registration, and the states provide legal authority and oversight, the implementation is usually decentralized to the state, county, or city level. Mosquito control operations are organized, in most instances, into fully independent mosquito abatement districts, public works departments, local health departments. In some cases, municipalities engage independent private contractors to undertake mosquito control within their jurisdictions. In sub-Saharan Africa (SSA), where most vector-borne disease endemic countries lie, mosquito control is organized centrally at the national level. In this model, the disease control programmes (national malaria control programmes or national malaria elimination programmes (NMCP/NMEP)) are embedded within the central governments' ministries of health (MoHs) and drive vector control policy development and implementation. Because of the high disease burden and limited resources, the primary endpoint of mosquito control in these settings is reduction of mosquito borne diseases, primarily, malaria. In the United States, however, the endpoint is mosquito control, therefore, significant (or even greater) emphasis is laid on nuisance mosquitoes as much as disease vectors. The authors detail experiences and learnings gathered by the delegation of African vector control professionals that participated in a formal exchange programme initiated by the Pan-African Mosquito Control Association (PAMCA), the University of Notre Dame, and members of the American Mosquito Control Association (AMCA), in the United States between the year 2021 and 2022. The authors highlight the key components of mosquito control operations in the United States and compare them to mosquito control programmes in SSA countries endemic for vector-borne diseases, deriving important lessons that could be useful for vector control in SSA.

A chromosomal reference genome sequence for the malaria mosquito, Anopheles moucheti, Evans, 1925.

We present a genome assembly from an individual male Anopheles moucheti (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), from a wild population in Cameroon. The genome sequence is 271 megabases in span. The majority of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.5 kilobases in length.

Evolution and spread of Plasmodium falciparum mutations associated with resistance to sulfadoxine-pyrimethamine in central Africa: a cross-sectional study.

BACKGROUND: Efficacy of sulfadoxine-pyrimethamine, the malaria chemoprophylaxis used in pregnant women, and in children when combined with amodiaquine, is threatened by the accumulation of mutations in the Plasmodium falciparum dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes. Data on the prevalence of resistant alleles in central Africa and the new pfdhps I431V mutation, particularly associated with other mutations to form the pfdhps vagKgs allele, are scarce. We explored the frequency and geographical distribution of pfdhps and pfdhfr mutations in central Africa in 2014-18, and assessed the evolutionary origin of the vagKgs allele. METHODS: Samples were collected at 18 health-care centres in seven countries (Angola, Cameroon, Central African Republic, Democratic Republic of the Congo, Gabon, Nigeria, and Republic of the Congo) from patients who showed possible symptoms of malaria between March 1, 2014, and Oct 31, 2018. Samples that were positive for P falciparum were transported to a laboratory in Toulouse, France, and genotyped. The frequency of pfdhfr and pfdhps mutations was studied in 1749 samples. Microsatellites in pfdhps flanking regions and whole-genome analysis compared with parasite genomes from the data-sharing network MalariaGEN were performed on samples carrying the vagKgs allele. FINDINGS: Mapping of the prevalence of single nucleotide polymorphisms and corresponding alleles of pfdhfr and pfdhps showed a substantial spread of alleles associated with sulfadoxine-pyrimethamine resistance in central Africa during the 2014-18 period, especially an increase going west to east in pfdhps alleles carrying the K540E and A581G mutations. A high prevalence of the pfdhps I431V mutation was observed in Cameroon (exceeding 50% in the northern region) and Nigeria. Genomic analysis showed a recent African emergence and a clonal expansion of the most frequent pfdhps vagKgs allele. INTERPRETATION: Reduced sulfadoxine-pyrimethamine efficacy due to increased resistance is a worrying situation, especially because the malaria transmission level is high in central Africa. Although the resistance phenotype remains to be confirmed, the emergence and spread of the vagKgs allele in west and central Africa could challenge the use of sulfadoxine-pyrimethamine. FUNDING: Toulouse Institute for Infectious and Inflammatory Diseases.

High Prevalence of Polyclonal Plasmodium falciparum Infections and Association with Poor IgG Antibody Responses in a Hyper-Endemic Area in Cameroon.

Malaria remains a major public health problem worldwide, with eradication efforts thwarted by drug and insecticide resistance and the lack of a broadly effective malaria vaccine. In continuously exposed communities, polyclonal infections are thought to reduce the risk of severe disease and promote the establishment of asymptomatic infections. We sought to investigate the relationship between the complexity of P. falciparum infection and underlying host adaptive immune responses in an area with a high prevalence of asymptomatic parasitaemia in Cameroon. A cross-sectional study of 353 individuals aged 2 to 86 years (median age = 16 years) was conducted in five villages in the Centre Region of Cameroon. Plasmodium falciparum infection was detected by multiplex nested PCR in 316 samples, of which 278 were successfully genotyped. Of these, 60.1% (167/278) were polyclonal infections, the majority (80.2%) of which were from asymptomatic carriers. Host-parasite factors associated with polyclonal infection in the study population included peripheral blood parasite density, participant age and village of residence. The number of parasite clones per infected sample increased significantly with parasite density (r = 0.3912, p < 0.0001) but decreased with participant age (r = -0.4860, p < 0.0001). Parasitaemia and the number of clones per sample correlated negatively with total plasma levels of IgG antibodies to three highly reactive P. falciparum antigens (MSP-1p19, MSP-3 and EBA175) and two soluble antigen extracts (merozoite and mixed stage antigens). Surprisingly, we observed no association between the frequency of polyclonal infection and susceptibility to clinical disease as assessed by the recent occurrence of malarial symptoms or duration since the previous fever episode. Overall, the data indicate that in areas with the high perennial transmission of P. falciparum, parasite polyclonality is dependent on underlying host antibody responses, with the majority of polyclonal infections occurring in persons with low levels of protective anti-plasmodial antibodies.

Effectiveness of Intermittent Preventive Treatment with Sulfadoxine-Pyrimethamine in Pregnancy: Low Coverage and High Prevalence of Plasmodium falciparum dhfr-dhps Quintuple Mutants as Major Challenges in Douala, an Urban Setting in Cameroon.

Intermittent preventive treatment in pregnancy with sulfadoxine and pyrimethamine (IPTp-SP) is a key component in the malaria control strategy implemented in Africa. The aim of this study was to determine IPTp-SP adherence and coverage, and the impact on maternal infection and birth outcomes in the context of widespread SP resistance in the city of Douala, Cameroon. Clinical and demographic information were documented among 888 pregnant women attending 3 health facilities, from the antenatal care visit to delivery. Positive samples were genotyped for P. falciparum gene (dhfr, dhps, and k13) mutations. The overall IPTp-SP coverage (≥three doses) was 17.5%, and 5.1% received no dose. P. falciparum prevalence was 16%, with a predominance of submicroscopic infections (89.3%). Malaria infection was significantly associated with locality and history of malaria, and it was reduced among women using indoor residual spraying. Optimal doses of IPTp-SP were significantly associated with reduced infection among newborns and women (secundiparous and multiparous), but there was no impact of IPTp-SP on the newborn bodyweight. Pfdhfr-Pfdhps quintuple mutants were over-represented (IRNI-FGKAA, IRNI-AGKAA), and sextuple mutants (IRNI-AGKAS, IRNI-FGEAA, IRNI-AGKGS) were also reported. The Pfk13 gene mutations associated with artemisinin resistance were not detected. This study highlights the role of ANC in achieving optimal SP coverage in pregnant women, the mitigated impact of IPTp-SP on malaria outcomes, and the high prevalence of multiple SP-resistant P. falciparum parasites in the city of Douala that could compromise the efficacy of IPTp-SP.

Influence of the sickle cell trait on Plasmodium falciparum infectivity from naturally infected gametocyte carriers.

BACKGROUND: Sickle cell trait (SCT) refers to the carriage of one abnormal copy of the ß-globin gene, the HbS allele. SCT offers protection against malaria, controlling parasite density and preventing progression to symptomatic malaria. However, it remains unclear whether SCT also affects transmission stages and mosquito infection parameters. Deciphering the impact of the SCT on human to mosquito malaria transmission is key to understanding mechanisms that maintain the trait in malaria endemic areas. METHODS: The study was conducted from June to July 2017 among asymptomatic children living in the locality of Mfou, Cameroon. Blood samples were collected from asymptomatic children to perform malaria diagnosis by microscopy, Plasmodium species by PCR and hemoglobin typing by RFLP. Infectiousness of gametocytes to mosquitoes was assessed by membrane feeding assays using blood from gametocyte carriers of HbAA and HbAS genotypes. A zero-inflated model was fitted to predict distribution of oocysts in mosquitoes according to hemoglobin genotype of the gametocyte source. RESULTS: Among the 1557 children enrolled in the study, 314 (20.16%) were of the HbAS genotype. The prevalence of children with P. falciparum gametocytes was 18.47% in HbAS individuals and 13.57% in HbAA, and the difference is significant (χ2 = 4.61, P = 0.032). Multiplicity of infection was lower in HbAS gametocyte carriers (median = 2 genotypes/carrier in HbAS versus 3.5 genotypes/carrier in HbAA, Wilcoxon sum rank test = 188, P = 0.032). Gametocyte densities in the blood donor significantly influenced mosquito infection prevalence in both HbAS and HbAA individuals. The HbAS genotype had no significant effect on mosquito infection outcomes when using immune or naïve serum in feeding assays. In AB replacement feeding experiments, the odds ratio of mosquito infection for HbAA blood as compared to HbAS was 0.56 (95% CI 0.29-1.10), indicating a twice higher risk of infection in mosquitoes fed on gametocyte-containing blood of HbAS genotype. CONCLUSION: Plasmodium transmission stages were more prevalent in SCT individuals. This may reflect the parasite's enhanced investment in the sexual stage to increase their survival rate when asexual replication is impeded. The public health impact of our results points the need for intensive malaria control interventions in areas with high prevalence of HbAS. The similar infection parameters in feeding experiments where mosquitoes received the original serum from the blood donor indicated that immune responses to gametocyte surface proteins occur in both HbAS and HbAA individuals. The higher risk of infection in mosquitoes fed on HbAS blood depleted of immune factors suggests that changes in the membrane properties in HbAS erythrocytes may impact on the maturation process of gametocytes within circulating red blood cells.

Genetic Diversity of Plasmodium falciparum and Distribution of Antimalarial Drug Resistance Mutations in Symptomatic and Asymptomatic Infections.

Malaria control relies on passive case detection, and this strategy fails detecting asymptomatic infections. In addition, infections in endemic areas harbor multiple parasite genotypes that could affect case management and malaria epidemiology. Here, we performed AmpSeq genotyping to capture polymorphisms associated with antimalarial resistance and the genetic diversity within natural Plasmodium falciparum infections. Known genetic polymorphisms associated with altered drug susceptibility were screened for the five most common marker genes, pfdhfr, pfdhps, pfmdr1, pfcrt, and pfK13, and genetic diversity was established from two known AmpSeq markers, cpmp and csp. Relative abundance of the different genotypes within mixed infections was calculated from the number of reads per genotype. Genotyping was performed on 117 samples, 63 from asymptomatic and 54 from symptomatic individuals. We identified up to 15 genotypes within an infection, and the median multiplicity of infection was higher in asymptomatic infections (median MOI = 5 in asymptomatics versus median MOI = 2 in symptomatics, P < 0.001). No genetic differentiation on parasites from asymptomatic and symptomatic individuals was found. No mutation associated with ART resistance was identified. Prevalence of the P. falciparum chloroquine resistance wild-type genotype (CVMNK) reached 80%, confirming a return to chloroquine (CQ) sensitive parasites in Cameroon. In addition, the CQ-associated resistant genotype (CVIET) was present at very low density in polyclonal infections. Persistence of low-density chloroquine resistant parasites indicates competition-survival trade-offs may contribute to maintaining genetic diversity in natura. Thus, monitoring the expansion of these low-density genotypes in different immune backgrounds will be critical to evaluate drug policy changes.

Transposable element variants and their potential adaptive impact in urban populations of the malaria vector Anopheles coluzzii.

Anopheles coluzzii is one of the primary vectors of human malaria in sub-Saharan Africa. Recently, it has spread into the main cities of Central Africa threatening vector control programs. The adaptation of An. coluzzii to urban environments partly results from an increased tolerance to organic pollution and insecticides. Some of the molecular mechanisms for ecological adaptation are known, but the role of transposable elements (TEs) in the adaptive processes of this species has not been studied yet. As a first step toward assessing the role of TEs in rapid urban adaptation, we sequenced using long reads six An. coluzzii genomes from natural breeding sites in two major Central Africa cities. We de novo annotated TEs in these genomes and in an additional high-quality An. coluzzii genome, and we identified 64 new TE families. TEs were nonrandomly distributed throughout the genome with significant differences in the number of insertions of several superfamilies across the studied genomes. We identified seven putatively active families with insertions near genes with functions related to vectorial capacity, and several TEs that may provide promoter and transcription factor binding sites to insecticide resistance and immune-related genes. Overall, the analysis of multiple high-quality genomes allowed us to generate the most comprehensive TE annotation in this species to date and identify several TE insertions that could potentially impact both genome architecture and the regulation of functionally relevant genes. These results provide a basis for future studies of the impact of TEs on the biology of An. coluzzii.

High Prevalence of Asymptomatic Malarial Anemia and Association with Early Conversion from Asymptomatic to Symptomatic Infection in a Plasmodium falciparum Hyperendemic Setting in Cameroon.

Asymptomatic malarial parasitemia is highly prevalent in Plasmodium falciparum endemic areas and often associated with increased prevalence of mild to moderate anemia. The aim of this study was to assess the prevalence of anemia during asymptomatic malaria parasitemia and its interplay with persistent infection in highly exposed individuals. A household-based longitudinal survey was undertaken in a malaria hyperendemic area in Cameroon using multiplex nested polymerase chain reaction to detect plasmodial infections. Residents with P. falciparum asymptomatic parasitemia were monitored over a 3-week period with the aid of structured questionnaires and weekly measurements of axillary temperatures. Of the 353 individuals included (median age: 26 years, range 2-86 years, male/female sex ratio 0.9), 328 (92.9%) were positive for malaria parasitemia of whom 266 (81.1%) were asymptomatic carriers. The prevalence of anemia in the study population was 38.6%, of which 69.2% were asymptomatic. Multivariate analyses identified high parasitemia (> 327 parasites/µL) and female gender as associated risk factors of asymptomatic malarial anemia in the population. Furthermore, risk analyses revealed female gender and anemia at the time of enrolment as key predictors of early development of febrile illness (< 3 weeks post enrolment) among the asymptomatic individuals. Together, the data reveal an extremely high prevalence of asymptomatic malaria parasitemia and anemia in the study area, unveiling for the first time the association of asymptomatic malarial anemia with early clinical conversion from asymptomatic to symptomatic infection. Furthermore, these findings underscore the negative impact of asymptomatic malaria parasitemia on individual health, necessitating the development of appropriate control and preventive measures.

The Rare, the Best: Spread of Antimalarial-Resistant Plasmodium falciparum Parasites by Anopheles Mosquito Vectors.

The emergence of resistance to antimalarials has prompted the steady switch to novel therapies for decades. Withdrawal of antimalarials, such as chloroquine in sub-Saharan Africa in the late 1990s, led to rapid declines in the prevalence of resistance markers after a few years, raising the possibility of reintroducing them for malaria treatment. Here, we provide evidence that the mosquito vector plays a crucial role in maintaining parasite genetic diversity. We followed the transmission dynamics of Plasmodium falciparum parasites through its vector in natural infections from gametocytes contained in the blood of asymptomatic volunteers until sporozoites subsequently developed in the mosquito salivary glands. We did not find any selection of the mutant or wild-type pfcrt 76 allele during development in the Anopheles mosquito vector. However, microsatellite genotyping indicated that minority genotypes were favored during transmission through the mosquito. The analysis of changes in the proportions of mutant and wild-type pfcrt 76 alleles showed that, regardless of the genotype, the less-represented allele in the gametocyte population was more abundant in mosquito salivary glands, indicating a selective advantage of the minority allele in the vector. Selection of minority genotypes in the vector would explain the persistence of drug-resistant alleles in the absence of drug pressure in areas with high malaria endemicity and high genetic diversity. Our results may have important epidemiological implications, as they predict the rapid re-emergence and spread of resistant genotypes if antimalarials that had previously selected resistant parasites are reintroduced for malaria prevention or treatment. IMPORTANCE Drug selection pressure in malaria patients is the cause of the emergence of resistant parasites. Resistance imposes a fitness cost for parasites in untreated infections, so withdrawal of the drug leads to the return of susceptible parasites. Little is known about the role of the malaria vector in this phenomenon. In an experimental study conducted in Cameroon, an area of high malaria transmission, we showed that the vector did not favor the parasites based on sensitivity or resistance criteria, but it did favor the selection of minority clones. This finding shows that the vector increases the diversity of plasmodial populations and could play an important role in falciparum malaria epidemiology by maintaining resistant clones despite the absence of therapeutic pressure.

Monoclonal antibodies block transmission of genetically diverse Plasmodium falciparum strains to mosquitoes.

Malaria parasite transmission to mosquitoes relies on the uptake of sexual stage parasites during a blood meal and subsequent formation of oocysts on the mosquito midgut wall. Transmission-blocking vaccines (TBVs) and monoclonal antibodies (mAbs) target sexual stage antigens to interrupt human-to-mosquito transmission and may form important tools for malaria elimination. Although most epitopes of these antigens are considered highly conserved, little is known about the impact of natural genetic diversity on the functional activity of transmission-blocking antibodies. Here we measured the efficacy of three mAbs against leading TBV candidates (Pfs48/45, Pfs25 and Pfs230) in transmission assays with parasites from naturally infected donors compared to their efficacy against the strain they were raised against (NF54). Transmission-reducing activity (TRA) was measured as reduction in mean oocyst intensity. mAb 45.1 (α-Pfs48/45) and mAb 4B7 (α-Pfs25) reduced transmission of field parasites from almost all donors with IC80 values similar to NF54. Sequencing of oocysts that survived high mAb concentrations did not suggest enrichment of escape genotypes. mAb 2A2 (α-Pfs230) only reduced transmission of parasites from a minority of the donors, suggesting that it targets a non-conserved epitope. Using six laboratory-adapted strains, we revealed that mutations in one Pfs230 domain correlate with mAb gamete surface binding and functional TRA. Our findings demonstrate that, despite the conserved nature of sexual stage antigens, minor sequence variation can significantly impact the efficacy of transmission-blocking mAbs. Since mAb 45.1 shows high potency against genetically diverse strains, our findings support its further clinical development and may inform Pfs48/45 vaccine design.

Antimalarial drug resistance in the Central and Adamawa regions of Cameroon: Prevalence of mutations in P. falciparum crt, Pfmdr1, Pfdhfr and Pfdhps genes.

The spread of Plasmodium falciparum resistant parasites remains one of the major challenges for malaria control and elimination in Sub Saharan Africa. Monitoring of molecular markers conferring resistance to different antimalarials is important to track the spread of resistant parasites and to optimize the therapeutic lifespan of current drugs. This study aimed to evaluate the prevalence of known mutations in the drug resistance genes Pfcrt, Pfmdr1, Pfdhfr and Pfdhps in two different epidemiological settings in Cameroon. Dried blood spots collected in 2018 and 2019 from asymptomatic individuals were used for DNA extraction and then the Plasmodium infection status was determined byPCR. Detection of SNPs was performed by nested PCR followed by allele-specific restriction analysis (ASRA). The prevalence of each genotype was compared between sites using the Chi square and Fisher's exact tests. A high prevalence of the Pfcrt K76 wild type allele was found in both sites (88.5 and 62.29% respectively; P< 0,0001). The prevalence of Pfmdr1 mutations 86Y and 1246Y was respectively 55.83 and 1.45% in Mfou and 45.87 and 5.97% in Tibati, with significant difference between the studied areas (P<0.0001). Overall, the Pfdhfr triple-mutant genotype (51I/59R/108N) was highly prevalent (> 96%), however no SNP was detected at codon 164. In Pfdhps, the prevalence of the 437G mutation reached (90%) and was at higher frequency in Mfou (P< 0.0001). Overall, the Pfdhps mutations 540E and 581G were less common (0.33 and 3.26%, respectively). The quadruple resistant genotype (Pfdhfr 51I/59R/108N+Pfdhp437G) was found almost 90% of the samples. The wild-type genotype (Pfdhfr N51/C59/S108/164I+Pfdhps A437/K540/A581) was never identified and the sextuple mutant (Pfdhfr 51I/59R/108N+Pfdhp437G/540E/581G), kwon as super resistant appeared in two samples from Tibati. These findings demonstrate declining trends in the prevalence of mutations conferring resistance to 4-aminoquinolines, especially to chloroquine. However, a high level of mutations in P. falciparum genes related to SP resistance was detected and this raises concerns about the future efficacy of IPTp-SP and SMC in Cameroon.

Efficacy of dihydroartemisinin/piperaquine in patients with non-complicated Plasmodium falciparum malaria in Yaoundé, Cameroon.

BACKGROUND: Dihydroartemisinin/piperaquine is increasingly used for the treatment of uncomplicated Plasmodium falciparum malaria in Africa. The efficacy of this combination in Cameroon is poorly documented, while resistance to dihydroartemisinin/piperaquine readily spreads in Southeast Asia. OBJECTIVES: This study evaluated the clinical efficacy of dihydroartemisinin/piperaquine in Cameroon, as well as the molecular profile and phenotypic susceptibility of collected isolates to dihydroartemisinin and piperaquine. PATIENTS AND METHODS: Dihydroartemisinin/piperaquine efficacy in 42 days was followed-up for 138 patients presenting non-complicated falciparum malaria. Piperaquine concentration was determined at day 7 for 124 patients. kelch13 gene polymorphisms (n = 150) and plasmepsin2 gene amplification (n = 148) were determined as molecular markers of resistance to dihydroartemisinin and piperaquine, respectively. Parasite susceptibility to dihydroartemisinin and piperaquine was determined using validated in vitro survival assays. RESULTS: The efficacy of dihydroartemisinin/piperaquine treatment was 100% after PCR correction. The reinfections were not associated with a variation of piperaquine concentration at day 7. Ninety-six percent (144/150) of the samples presented a WT allele of the kelch13 gene. Two percent (3/150) presented the non-synonymous mutation A578S, which is not associated with resistance to dihydroartemisinin. No duplication of the plasmepsin2 gene was observed (0/148). All the samples tested in vitro by survival assays (n = 87) were susceptible to dihydroartemisinin and piperaquine. CONCLUSIONS: Dihydroartemisinin/piperaquine has demonstrated excellent therapeutic efficacy with no evidence of emerging artemisinin or piperaquine resistance in Yaoundé, Cameroon. This observation suggests that dihydroartemisinin/piperaquine could be a sustainable therapeutic solution for P. falciparum malaria if implemented in areas previously free of artemisinin- and piperaquine-resistant parasites, unlike Southeast Asia.

Epidemiological and entomological studies of malaria transmission in Tibati, Adamawa region of Cameroon 6 years following the introduction of long-lasting insecticide nets.

BACKGROUND: Malaria remains a serious public health problem in Cameroon. Implementation of control interventions requires prior knowledge of the local epidemiological situation. Here we report the results of epidemiological and entomological surveys carried out in Tibati, Adamawa Region, Cameroon, an area where malaria transmission is seasonal, 6 years after the introduction of long-lasting insecticidal bed nets. METHODS: Cross-sectional studies were carried out in July 2015 and 2017 in Tibati. Thick blood smears and dried blood spots were collected from asymptomatic and symptomatic individuals in the community and at health centers, respectively, and used for the molecular diagnosis of Plasmodium species. Adult mosquitoes were collected by indoor residual spraying and identified morphologically and molecularly. The infection status of Plasmodium spp. was determined by quantitative PCR, and positivity of PCR-positive samples was confirmed by Sanger sequencing. RESULTS: Overall malaria prevalence in our study population was 55.0% (752/1367) and Plasmodium falciparum was the most prevalent parasite species (94.3%), followed by P. malariae (17.7%) and P. ovale (0.8%); 92 (12.7%) infections were mixed infections. Infection parameters varied according to clinical status (symptomatic/asymptomatic) and age of the sampled population and the collection sites. Infection prevalence was higher in asymptomatic carriers (60.8%), but asexual and sexual parasite densities were lower. Prevalence and intensity of infection decreased with age in both the symptomatic and asymptomatic groups. Heterogeneity in infections was observed at the neighborhood level, revealing hotspots of transmission. Among the 592 Anopheles mosquitoes collected, 212 (35.8%) were An. gambiae, 172 (29.1%) were An. coluzzii and 208 (35.1%) were An. funestus (s.s.). A total of 26 (4.39%) mosquito specimens were infected by Plasmodium sp. and the three Anopheles mosquitoes transmitted Plasmodium at equal efficiency. Surprisingly, we found an An. coluzzii specimen infected by Plasmodium vivax, which confirms circulation of this species in Cameroon. The positivity of all 26 PCR-positive Plasmodium-infected mosquitoes was successively confirmed by sequencing analysis. CONCLUSION: Our study presents the baseline malaria parasite burden in Tibati, Adamawa Region, Cameroon. Our results highlight the high malaria endemicity in the area, and hotspots of disease transmission are identified. Parasitological indices suggest low bednet usage and that implementation of control interventions in the area is needed to reduce malaria burden. We also report for the first time a mosquito vector with naturally acquired P. vivax infection in Cameroon.

Immunofocusing humoral immunity potentiates the functional efficacy of the AnAPN1 malaria transmission-blocking vaccine antigen.

Malaria transmission-blocking vaccines (TBVs) prevent the completion of the developmental lifecycle of malarial parasites within the mosquito vector, effectively blocking subsequent infections. The mosquito midgut protein Anopheline alanyl aminopeptidase N (AnAPN1) is the leading, mosquito-based TBV antigen. Structure-function studies identified two Class II epitopes that can induce potent transmission-blocking (T-B) antibodies, informing the design of the next-generation AnAPN1. Here, we functionally screened new immunogens and down-selected to the UF6b construct that has two glycine-linked copies of the T-B epitopes. We then established a process for manufacturing UF6b and evaluated in outbred female CD1 mice the immunogenicity of the preclinical product with the human-safe adjuvant Glucopyranosyl Lipid Adjuvant in a liposomal formulation with saponin QS21 (GLA-LSQ). UF6b:GLA-LSQ effectively immunofocused the humoral response to one of the key T-B epitopes resulting in potent T-B activity, underscoring UF6b as a prime TBV candidate to aid in malaria elimination and eradication efforts.

Preliminary validation of the use of IgG antibody response to Anopheles gSG6-p1 salivary peptide to assess human exposure to malaria vector bites in two endemic areas of Cameroon in Central Africa.

The specific immune response to the Anopheles salivary peptide could be a pertinent and complementary tool to assess the risk of malaria transmission and the effectiveness of vector control strategies. This study aimed to obtain first reliable data on the current state of the Anopheles gSG6-P1 biomarker for assess the level of exposure to Anopheles bites in high malaria endemic areas in Cameroon. Blood smears were collected from people living in the neighborhoods of Youpwe (suburban area, continental) and Manoka (rural area, Island), both areas in the coastal region of Cameroon. Malaria infection was determined using thick blood smear microscopy, whereas the level of specific IgG response to gSG-P1 peptide was assessed by enzyme-linked immunosorbent assay from the dried blood spots. Of 266 (153 from Youpwe, 113 from Manoka) malaria endemic residents (mean age: 22.8±19.8 years, age range: 6 months-94 years, male/female sex ratio: 1/1.2, with Manoka mean age: 23.71±20.53, male/female sex ratio:1/1.13 and Youpwe mean age: 22.12±19.22, male/female sex ratio 1/0.67) randomly included in the study, Plasmodium infection prevalence was significantly higher in Manoka than in Youpwe (64.6% vs 12,4%, p = 0.0001). The anti-gSG6-P1 IgG response showed a high inter-individual heterogeneity and was significantly higher among individuals from Manoka than those from Youpwe (p = 0.023). Malaria infected individuals presented a higher anti-gSG6-P1 IgG antibody response than non-infected (p = 0.0004). No significant difference in the level of specific IgG response to gSG-P1 was observed according to long lasting insecticidal nets use. Taken together, the data revealed that human IgG antibody response to Anopheles gSG-P1 salivary peptide could be also used to assess human exposure to malaria vectors in Central African region. This finding strengthens the relevance of this candidate biomarker to be used for measuring human exposure to malaria vectors worldwide.

K-13 propeller gene polymorphisms isolated between 2014 and 2017 from Cameroonian Plasmodium falciparum malaria patients.

The emergence of artemisinin-resistant parasites since the late 2000s at the border of Cambodia and Thailand poses serious threats to malaria control globally, particularly in Africa which bears the highest malaria transmission burden. This study aimed to obtain reliable data on the current state of the kelch13 molecular marker for artemisinin resistance in Plasmodium falciparum in Cameroon. DNA was extracted from the dried blood spots collected from epidemiologically distinct endemic areas in the Center, Littoral and North regions of Cameroon. Nested PCR products from the Kelch13-propeller gene were sequenced and analyzed on an ABI 3730XL automatic sequencer. Of 219 dried blood spots, 175 were sequenced successfully. We identified six K13 mutations in 2.9% (5/175) of samples, including 2 non-synonymous, the V589I allele had been reported in Africa already and one new allele E612K had not been reported yet. These two non-synonymous mutations were uniquely found in parasites from the Littoral region. One sample showed two synonymous mutations within the kelch13 gene. We also observed two infected samples with mixed K13 mutant and K13 wild-type infection. Taken together, our data suggested the circulation of the non-synonymous K13 mutations in Cameroon. Albeit no mutations known to be associated with parasite clearance delays in the study population, there is need for continuous surveillance for earlier detection of resistance as long as ACTs are used and scaled up in the community.

Mosquito microevolution drives Plasmodium falciparum dynamics.

Malaria, a major cause of child mortality in Africa, is engendered by Plasmodium parasites that are transmitted by anopheline mosquitoes. Fitness of Plasmodium parasites is closely linked to the ecology and evolution of its anopheline vector. However, whether the genetic structure of vector populations impacts malaria transmission remains unknown. Here, we describe a partitioning of the African malaria vectors into generalists and specialists that evolve along ecological boundaries. We next identify the contribution of mosquito species to Plasmodium abundance using Granger causality tests for time-series data collected over two rainy seasons in Mali. We find that mosquito microevolution, defined by changes in the genetic structure of a population over short ecological timescales, drives Plasmodium dynamics in nature, whereas vector abundance, infection prevalence, temperature and rain have low predictive values. Our study demonstrates the power of time-series approaches in vector biology and highlights the importance of focusing local vector control strategies on mosquito species that drive malaria dynamics.

A saliva-based rapid test to quantify the infectious subclinical malaria parasite reservoir.

A large proportion of ongoing malaria parasite transmission is attributed to low-density subclinical infections not readily detected by available rapid diagnostic tests (RDTs) or microscopy. Plasmodium falciparum gametocyte carriage is subclinical, but gametocytemic individuals comprise the parasite reservoir that leads to infection of mosquitoes and local transmission. Effective detection and quantification of these carriers can help advance malaria elimination strategies. However, no point-of-need (PON) RDTs for gametocyte detection exist, much less one that can perform noninvasive sampling of saliva outside a clinical setting. Here, we report on the discovery of 35 parasite markers from which we selected a single candidate for use in a PON RDT. We performed a cross-sectional, multi-omics study of saliva from 364 children with subclinical infection in Cameroon and Zambia and produced a prototype saliva-based PON lateral flow immunoassay test for P. falciparum gametocyte carriers. The test is capable of identifying submicroscopic carriage in both clinical and nonclinical settings and is compatible with archived saliva samples.