Parallel Evolution in Mosquito Vectors-A Duplicated Esterase Locus is Associated With Resistance to Pirimiphos-methyl in Anopheles gambiae.

The primary control methods for the African malaria mosquito, Anopheles gambiae, are based on insecticidal interventions. Emerging resistance to these compounds is therefore of major concern to malaria control programs. The organophosphate (OP), pirimiphos-methyl, is a relatively new chemical in the vector control armory but is now widely used in indoor-residual spray campaigns. While generally effective, phenotypic resistance has developed in some areas in malaria vectors. Here, we used a population genomic approach to identify novel mechanisms of resistance to pirimiphos-methyl in A. gambiae s.l mosquitoes. In multiple populations, we found large and repeated signals of selection at a locus containing a cluster of detoxification enzymes, some of whose orthologs are known to confer resistance to OPs in Culex pipiens. Close examination revealed a pair of alpha-esterases, Coeae1f and Coeae2f, and a complex and diverse pattern of haplotypes under selection in A. gambiae, A. coluzzii and A. arabiensis. As in C. pipiens, copy number variants have arisen at this locus. We used diplotype clustering to examine whether these signals arise from parallel evolution or adaptive introgression. Using whole-genome sequenced phenotyped samples, we found that in West Africa, a copy number variant in A. gambiae is associated with resistance to pirimiphos-methyl. Overall, we demonstrate a striking example of contemporary parallel evolution which has important implications for malaria control programs.

Transcriptomic analysis of Anopheles gambiae from Benin reveals overexpression of salivary and cuticular proteins associated with cross-resistance to pyrethroids and organophosphates.

BACKGROUND: Insecticide resistance (IR) is one of the major threats to malaria vector control programs in endemic countries. However, the mechanisms underlying IR are poorly understood. Thus, investigating gene expression patterns related to IR can offer important insights into the molecular basis of IR in mosquitoes. In this study, RNA-Seq was used to characterize gene expression in Anopheles gambiae surviving exposure to pyrethroids (deltamethrin, alphacypermethrin) and an organophosphate (pirimiphos-methyl). RESULTS: Larvae of An. gambiae s.s. collected from Bassila and Djougou in Benin were reared to adulthood and phenotyped for IR using a modified CDC intensity bottle bioassay. The results showed that mosquitoes from Djougou were more resistant to pyrethroids (5X deltamethrin: 51.7% mortality; 2X alphacypermethrin: 47.4%) than Bassila (1X deltamethrin: 70.7%; 1X alphacypermethrin: 77.7%), while the latter were more resistant to pirimiphos-methyl (1.5X: 48.3% in Bassila and 1X: 21.5% in Djougou). RNA-seq was then conducted on resistant mosquitoes, non-exposed mosquitoes from the same locations and the laboratory-susceptible An. gambiae s.s. Kisumu strain. The results showed overexpression of detoxification genes, including cytochrome P450s (CYP12F2, CYP12F3, CYP4H15, CYP4H17, CYP6Z3, CYP9K1, CYP4G16, and CYP4D17), carboxylesterase genes (COEJHE5E, COE22933) and glutathione S-transferases (GSTE2 and GSTMS3) in all three resistant mosquito groups analyzed. Genes encoding cuticular proteins (CPR130, CPR10, CPR15, CPR16, CPR127, CPAP3-C, CPAP3-B, and CPR76) were also overexpressed in all the resistant groups, indicating their potential role in cross resistance in An. gambiae. Salivary gland protein genes related to 'salivary cysteine-rich peptide' and 'salivary secreted mucin 3' were also over-expressed and shared across all resistant groups. CONCLUSION: Our results suggest that in addition to metabolic enzymes, cuticular and salivary gland proteins could play an important role in cross-resistance to multiple classes of insecticides in Benin. These genes warrant further investigation to validate their functional role in An. gambiae resistance to insecticides.

Key gene modules and hub genes associated with pyrethroid and organophosphate resistance in Anopheles mosquitoes: a systems biology approach.

Indoor residual spraying (IRS) and insecticide-treated nets (ITNs) are the main methods used to control mosquito populations for malaria prevention. The efficacy of these strategies is threatened by the spread of insecticide resistance (IR), limiting the success of malaria control. Studies of the genetic evolution leading to insecticide resistance could enable the identification of molecular markers that can be used for IR surveillance and an improved understanding of the molecular mechanisms associated with IR. This study used a weighted gene co-expression network analysis (WGCNA) algorithm, a systems biology approach, to identify genes with similar co-expression patterns (modules) and hub genes that are potential molecular markers for insecticide resistance surveillance in Kenya and Benin. A total of 20 and 26 gene co-expression modules were identified via average linkage hierarchical clustering from Anopheles arabiensis and An. gambiae, respectively, and hub genes (highly connected genes) were identified within each module. Three specific genes stood out: serine protease, E3 ubiquitin-protein ligase, and cuticular proteins, which were top hub genes in both species and could serve as potential markers and targets for monitoring IR in these malaria vectors. In addition to the identified markers, we explored molecular mechanisms using enrichment maps that revealed a complex process involving multiple steps, from odorant binding and neuronal signaling to cellular responses, immune modulation, cellular metabolism, and gene regulation. Incorporation of these dynamics into the development of new insecticides and the tracking of insecticide resistance could improve the sustainable and cost-effective deployment of interventions.

DNA Nanostructure-Templated Antibody Complexes Provide Insights into the Geometric Requirements of Human Complement Cascade Activation.

The classical complement pathway is activated by antigen-bound IgG antibodies. Monomeric IgG must oligomerize to activate complement via the hexameric C1q complex, and hexamerizing mutants of IgG appear as promising therapeutic candidates. However, structural data have shown that it is not necessary to bind all six C1q arms to initiate complement, revealing a symmetry mismatch between C1 and the hexameric IgG complex that has not been adequately explained. Here, we use DNA nanotechnology to produce specific nanostructures to template antigens and thereby spatially control IgG valency. These DNA-nanotemplated IgG complexes can activate complement on cell-mimetic lipid membranes, which enabled us to determine the effect of IgG valency on complement activation without the requirement to mutate antibodies. We investigated this using biophysical assays together with 3D cryo-electron tomography. Our data revealed the importance of interantigen distance on antibody-mediated complement activation, and that the cleavage of complement component C4 by the C1 complex is proportional to the number of ideally spaced antigens. Increased IgG valency also translated to better terminal pathway activation and membrane attack complex formation. Together, these data provide insights into how nanopatterning antigen-antibody complexes influence the activation of the C1 complex and suggest routes to modulate complement activation by antibody engineering. Furthermore, to our knowledge, this is the first time DNA nanotechnology has been used to study the activation of the complement system.

Vectorial competence, insecticide resistance in Anopheles funestus and operational implications for malaria vector control strategies in Benin Republic.

The primary reason for the failure of malaria vector control across endemic regions is the widespread insecticide resistance observed in Anopheles vectors. The most dominant African vectors of malaria parasites are Anopheles gambiae and Anopheles funestus mosquitoes. These species often exhibit divergent behaviours and adaptive changes underscoring the importance of deploying active and effective measures in their control. Unlike An. gambiae, An. funestus mosquitoes are poorly studied in Benin Republic. However, recent reports indicated that An. funestus can adapt and colonize various ecological niches owing to its resistance against insecticides and adaptation to changing breeding habitats. Unfortunately, scientific investigations on the contribution of An. funestus to malaria transmission, their susceptibility to insecticide and resistance mechanism developed are currently insufficient for the design of better control strategies. In an attempt to gather valuable information on An. funestus, the present review examines the progress made on this malaria vector species in Benin Republic and highlights future research perspectives on insecticide resistance profiles and related mechanisms, as well as new potential control strategies against An. funestus. Literature analysis revealed that An. funestus is distributed all over the country, although present in low density compared to other dominant malaria vectors. Interestingly, An. funestus is being found in abundance during the dry seasons, suggesting an adaptation to desiccation. Among the An. funestus group, only An. funestus sensu stricto (s.s.) and Anopheles leesoni were found in the country with An. funestus s.s. being the most abundant species. Furthermore, An. funestus s.s. is the only one species in the group contributing to malaria transmission and have adapted biting times that allow them to bite at dawn. In addition, across the country, An. funestus were found resistant to pyrethroid insecticides used for bed nets impregnation and also resistant to bendiocarb which is currently being introduced in indoor residual spraying formulation in malaria endemic regions. All these findings highlight the challenges faced in controlling this malaria vector. Therefore, advancing the knowledge of vectorial competence of An. funestus, understanding the dynamics of insecticide resistance in this malaria vector, and exploring alternative vector control measures, are critical for sustainable malaria control efforts in Benin Republic.

Relative expression of key genes involved in nucleic acids methylation in Anopheles gambiae sensu stricto.

In vertebrates, enzymes responsible for DNA methylation, one of the epigenetic mechanisms, are encoded by genes falling into the cytosine methyltransferases genes family (Dnmt1, Dnmt3a,b and Dnmt3L). However, in Diptera, only the methyltransferase Dnmt2 was found, suggesting that DNA methylation might act differently for species in this order. Moreover, genes involved in epigenetic dynamics, such as Ten-eleven Translocation dioxygenases (TET) and Methyl-CpG-binding domain (MBDs), present in vertebrates, might play a role in insects. This work aimed at investigating nucleic acids methylation in the malaria vector Anopheles gambiae (Diptera: Culicidae) by analysing the expression of Dnmt2, TET2 and MBDs genes using quantitative real-time polymerase chain reaction (qRT-PCR) at pre-immature stages and in reproductive tissues of adult mosquitoes. In addition, the effect of two DNA methylation inhibitors on larval survival was evaluated. The qPCR results showed an overall low expression of Dnmt2 at all developmental stages and in adult reproductive tissues. In contrast, MBD and TET2 showed an overall higher expression. In adult mosquito reproductive tissues, the expression level of the three genes in males' testes was significantly higher than that in females' ovaries. The chemical treatments did not affect larval survival. The findings suggest that mechanisms other than DNA methylation underlie epigenetic regulation in An. gambiae.

Surveillance of Plasmodium malariae infection among inhabitants of rural areas in Ouidah-Kpomasse-Tori Bossito health district, Benin.

Among the Plasmodium species that infect humans, P. falciparum has been largely studied in malaria endemic areas. However, P. malariae infection is less documented among the human population. This study aimed to monitor the prevalence and distribution of P. malariae in Southern Benin. A cross-sectional survey was conducted in rural localities in the Ouidah-Kpomasse-Tori Bossito (OKT) health district in Southern Benin from June to October 2019. Socio-demographic data were collected using a questionnaire, while malaria infection data were obtained on the one hand by microscopy diagnosis and, on the other, by nested polymerase chain reaction (PCR). Based on microscopy, the prevalence of P. malariae mono-infection and coinfection of P. falciparum, P. malariae was respectively 2.3% and 1.2% in the OKT health district. This prevalence was higher (P < 0.01) than that reported by Damien et al. (2010) 10 years ago in the same study area with 0.7% and 0.3% of P. malariae and P. falciparum/P. malariae, respectively. Based on PCR analysis, P. malariae prevalence was 14.1%, including 5.2% of mono-infection and 8.9% of mixed infection with P. falciparum. Sub-microscopic Plasmodium infections were high (30.6%) and more pronounced in older participants (>20 years). The present study revealed that P. malariae increased in the OKT health district with a high prevalence of submicroscopic infection. Since our results provide valuable evidence of increasing P. malariae infection, the National Malaria Control Programs (NMCPs) must consider P. malariae when designing future measures for effective control and malaria treatment.

Putative pleiotropic effects of the knockdown resistance (L1014F) allele on the life-history traits of Anopheles gambiae.

BACKGROUND: Existing mechanisms of insecticide resistance are known to help the survival of mosquitoes following contact with chemical compounds, even though they could negatively affect the life-history traits of resistant malaria vectors. In West Africa, the knockdown resistance mechanism kdrR (L1014F) is the most common. However, little knowledge is available on its effects on mosquito life-history traits. The fitness effects associated with this knockdown resistance allele in Anopheles gambiae sensu stricto (s.s.) were investigated in an insecticide-free laboratory environment. METHODS: The life-history traits of Kisumu (susceptible) and KisKdr (kdr resistant) strains of An. gambiae s.s. were compared. Larval survivorship and pupation rate were assessed as well as fecundity and fertility of adult females. Female mosquitoes of both strains were directly blood fed through artificial membrane assays and then the blood-feeding success, blood volume and adult survivorship post-blood meal were assessed. RESULTS: The An. gambiae mosquitoes carrying the kdrR allele (KisKdr) laid a reduced number of eggs. The mean number of larvae in the susceptible strain Kisumu was three-fold overall higher than that seen in the KisKdr strain with a significant difference in hatching rates (81.89% in Kisumu vs 72.89% in KisKdr). The KisKdr larvae had a significant higher survivorship than that of Kisumu. The blood-feeding success was significantly higher in the resistant mosquitoes (84%) compared to the susceptible ones (34.75%). However, the mean blood volume was 1.36 µL/mg, 1.45 µL/mg and 1.68 µL/mg in Kisumu, homozygote and heterozygote KisKdr mosquitoes, respectively. After blood-feeding, the heterozygote KisKdr mosquitoes displayed highest survivorship when compared to that of Kisumu. CONCLUSIONS: The presence of the knockdown resistance allele appears to impact the life-history traits, such as fecundity, fertility, larval survivorship, and blood-feeding behaviour in An. gambiae. These data could help to guide the implementation of more reliable strategies for the control of malaria vectors.

Interplay Between Oxytetracycline and the Homozygote kdr (L1014F) Resistance Genotype on Fecundity in Anopheles gambiae (Diptera: Culicidae) Mosquitoes.

The insecticide resistance in Anopheles gambiae mosquitoes has remained the major threat for vector control programs but the fitness effects conferred by these mechanisms are poorly understood. To fill this knowledge gap, the present study aimed at testing the hypothesis that antibiotic oxytetracycline could have an interaction with insecticide resistance genotypes and consequently inhibit the fecundity in An. gambiae. Four strains of An. gambiae: Kisumu (susceptible), KisKdr (kdr (L1014F) resistant), AcerKis (ace-1 (G119S) resistant) and AcerKdrKis (both kdr (L1014F) and ace-1 (G119S) resistant) were used in this study. The different strains were allowed to bloodfeed on a rabbit previously treated with antibiotic oxytetracycline at a concentration of 39·10-5 M. Three days later, ovarian follicles were dissected from individual mosquito ovaries into physiological saline solution (0.9% NaCl) under a stereomicroscope and the eggs were counted. Fecundity was substantially lower in oxytetracycline-exposed KisKdr females when compared to that of the untreated individuals and oxytetracycline-exposed Kisumu females. The exposed AcerKis females displayed an increased fecundity compared to their nontreated counterparts whereas they had reduced fecundity compared to that of oxytetracycline-exposed Kisumu females. There was no substantial difference between the fecundity in the treated and untreated AcerKdrKis females. The oxytetracycline-exposed AcerKdrKis mosquitoes had an increased fecundity compared to that of the exposed Kisumu females. Our data indicate an indirect effect of oxytetracycline in reducing fecundity of An. gambiae mosquitoes carrying kdrR (L1014F) genotype. These findings could be useful for designing new integrated approaches for malaria vector control in endemic countries.

Performance of basophil activation test and specific IgG4 as diagnostic tools in nonspecific lipid transfer protein allergy: Antwerp-Barcelona comparison.

BACKGROUND: Recent studies show that nsLTP sensitization is not limited to the Mediterranean basin and can present diverse clinical phenotypes. It remains challenging to predict clinical outcome when specific IgE antibodies (sIgE) to nsLTPs are present. This study compares both clinical and in vitro allergy characteristics but also diagnostic performance of a basophil activation test (BAT) and sIgG4 in nsLTP-sensitized patients from Antwerp (ANT, Belgium) and Barcelona (BCN, Spain). METHODS: Adult subjects with positive sIgE rPru p 3 and/or rMal d 3 ≥ 0.10 kUA /L (n = 182) and healthy controls (n = 37) were included. NsLTP-sensitized individuals were stratified according to clinical symptoms with peach/apple, respectively. BAT rPru p 3 and rMal d 3 were performed and sIgG4 antibodies to both components quantified. RESULTS: In BCN, only ratios of sIgG4/sIgE rMal d 3 and BAT rMal d 3 (0.001 µg/mL) can identify clinically relevant Mal d 3 sensitization (sensitivity of 60%-63% and a specificity of 75%-67%, respectively). In ANT, only the sIgE/total IgE rPru p 3 ratio shows added value (sensitivity 60% and specificity 83%). Finally, it appears that symptomatic patients in BCN are more sensitive to lower allergen concentrations compared to ANT. In addition, it was shown that ANT patients were more often sensitized to pollen and that specific pollen sources differed between regions. CONCLUSIONS: NsLTP-related allergy profiles and diagnostic performance differ significantly between regions and are component-specific, which makes extrapolation of data difficult to do. In addition, it seems that basophil sensitivity might show geographical differences. Additional research is needed to confirm these findings.

The ace-1 Locus Is Amplified in All Resistant Anopheles gambiae Mosquitoes: Fitness Consequences of Homogeneous and Heterogeneous Duplications.

Gene copy-number variations are widespread in natural populations, but investigating their phenotypic consequences requires contemporary duplications under selection. Such duplications have been found at the ace-1 locus (encoding the organophosphate and carbamate insecticides' target) in the mosquito Anopheles gambiae (the major malaria vector); recent studies have revealed their intriguing complexity, consistent with the involvement of various numbers and types (susceptible or resistant to insecticide) of copies. We used an integrative approach, from genome to phenotype level, to investigate the influence of duplication architecture and gene-dosage on mosquito fitness. We found that both heterogeneous (i.e., one susceptible and one resistant ace-1 copy) and homogeneous (i.e., identical resistant copies) duplications segregated in field populations. The number of copies in homogeneous duplications was variable and positively correlated with acetylcholinesterase activity and resistance level. Determining the genomic structure of the duplicated region revealed that, in both types of duplication, ace-1 and 11 other genes formed tandem 203kb amplicons. We developed a diagnostic test for duplications, which showed that ace-1 was amplified in all 173 resistant mosquitoes analyzed (field-collected in several African countries), in heterogeneous or homogeneous duplications. Each type was associated with different fitness trade-offs: heterogeneous duplications conferred an intermediate phenotype (lower resistance and fitness costs), whereas homogeneous duplications tended to increase both resistance and fitness cost, in a complex manner. The type of duplication selected seemed thus to depend on the intensity and distribution of selection pressures. This versatility of trade-offs available through gene duplication highlights the importance of large mutation events in adaptation to environmental variation. This impressive adaptability could have a major impact on vector control in Africa.

Contemporary evolution of resistance at the major insecticide target site gene Ace-1 by mutation and copy number variation in the malaria mosquito Anopheles gambiae.

Functionally constrained genes are ideal insecticide targets because disruption is often fatal, and resistance mutations are typically costly. Synaptic acetylcholinesterase (AChE) is an essential neurotransmission enzyme targeted by insecticides used increasingly in malaria control. In Anopheles and Culex mosquitoes, a glycine-serine substitution at codon 119 of the Ace-1 gene confers both resistance and fitness costs, especially for 119S/S homozygotes. G119S in Anopheles gambiae from Accra (Ghana) is strongly associated with resistance, and, despite expectations of cost, resistant 119S alleles are increasing significantly in frequency. Sequencing of Accra females detected only a single Ace-1 119S haplotype, whereas 119G diversity was high overall but very low at non-synonymous sites, evidence of strong purifying selection driven by functional constraint. Flanking microsatellites showed reduced diversity, elevated linkage disequilibrium and high differentiation of 119S, relative to 119G homozygotes across up to two megabases of the genome. Yet these signals of selection were inconsistent and sometimes weak tens of kilobases from Ace-1. This unexpected finding is attributable to apparently ubiquitous amplification of 119S alleles as part of a large copy number variant (CNV) far exceeding the size of the Ace-1 gene, whereas 119G alleles were unduplicated. Ace-1 CNV was detectable in archived samples collected when the 119S allele was rare in Ghana. Multicopy amplification of resistant alleles has not been observed previously and is likely to underpin the recent increase in 119S frequency. The large CNV compromised localization of the strong selective sweep around Ace-1, emphasizing the need to integrate CNV analysis into genome scans for selection.

Estimation of allele-specific Ace-1 duplication in insecticide-resistant Anopheles mosquitoes from West Africa.

BACKGROUND: Identification of variation in Ace-1 copy number and G119S mutation genotype from samples of Anopheles gambiae and Anopheles coluzzii across West Africa are important diagnostics of carbamate and organophosphate resistance at population and individual levels. The most widespread and economical method, PCR-RFLP, suffers from an inability to discriminate true heterozygotes from heterozygotes with duplication. METHODS: In addition to PCR-RFLP, in this study three different molecular techniques were applied on the same mosquito specimens: TaqMan qPCR, qRTPCR and ddPCR. To group heterozygous individuals recorded from the PCR-RFLP analysis into different assumptive genotypes K-means clustering was applied on the Z-scores of data obtained from both the TaqMan and ddPCR methods. The qRTPCR analysis was used for absolute quantification of copy number variation. RESULTS: The results indicate that most heterozygotes are duplicated and that G119S mutation must now be regarded as a complex genotype ranging from primarily single-copy susceptible Glycine homozygotes to balanced and imbalanced heterozygotes, and multiply-amplified resistant Serine allele homozygotes. Whilst qRTPCR-based gene copy analysis suffers from some imprecision, it clearly illustrates differences in copy number among genotype groups identified by TaqMan or ddPCR. Based on TaqMan method properties, and by coupling TaqMan and ddPCR methods simultaneously on the same type of mosquito specimens, it demonstrated that the TaqMan genotype assays associated with the K-means clustering algorithm could provide a useful semi-quantitative estimate method to investigate the level of allele-specific duplication in mosquito populations. CONCLUSIONS: Ace-1 gene duplication is evidently far more complex in An. gambiae and An. coluzzii than the better-studied mosquito Culex quinquefasciatus, which consequently can no longer be considered an appropriate model for prediction of phenotypic consequences. These require urgent further evaluation in Anopheles. To maintain the sustained effectiveness carbamates and organophosphates as alternative products to pyrethroids for malaria vector control, monitoring of duplicated resistant alleles in natural populations is essential to guide the rational use of these insecticides.

Endogenous pain modulation in response to exercise in patients with rheumatoid arthritis, patients with chronic fatigue syndrome and comorbid fibromyalgia, and healthy controls: a double-blind randomized controlled trial.

OBJECTIVE: Temporal summation (TS) of pain, conditioned pain modulation (CPM), and exercise-induced analgesia (EIA) are often investigated in chronic pain populations as an indicator for enhanced pain facilitation and impaired endogenous pain inhibition, respectively, but interactions are not yet clear both in healthy controls and in chronic pain patients. Therefore, the present double-blind randomized placebo-controlled study evaluates pains cores, TS, and CPM in response to exercise in healthy controls, patients with chronic fatigue syndrome and comorbid fibromyalgia (CFS/FM), and patients with rheumatoid arthritis (RA), both under placebo and paracetamol condition. METHODS: Fifty-three female volunteers - of which 19 patients with CFS/FM, 16 patients with RA, and 18 healthy controls - underwent a submaximal exercise test on a bicycle ergometer on 2 different occasions (paracetamol vs. placebo), with an interval of 7 days. Before and after exercise, participants rated pain intensity during TS and CPM. RESULTS: Patients with rheumatoid arthritis showed decreased TS after exercise, both after paracetamol and placebo (P < 0.05). In patients with CFS/FM, results were less univocal. A nonsignificant decrease in TS was only observed after taking paracetamol. CPM responses to exercise are inconclusive, but seem to worsen after exercise. No adverse effects were seen. CONCLUSION: This study evaluates pain scores, TS, and CPM in response to submaximal exercise in 2 different chronic pain populations and healthy controls. In patients with RA, exercise had positive effects on TS, suggesting normal EIA. In patients with CFS/FM, these positive effects were only observed after paracetamol and results were inconsistent.

Cor a 14: missing link in the molecular diagnosis of hazelnut allergy?

BACKGROUND: Hazelnut allergy shows distinct clinical patterns that can be predicted through component-resolved diagnosis. However, identification of sensitization profiles remains incomplete. METHODS: Sera of 75 patients allergic to hazelnuts, 14 infants with atopic dermatitis (AD) sensitized to hazelnuts, 15 hazelnut-tolerant individuals with specific IgE (sIgE) to hazelnuts and 15 healthy control individuals were tested for sIgE reactivity to rCor a 1.04, rCor a 8, nCor a 9, nCor a 11, rCor a 14, rBet v 1, rBet v 2 and cross-reactive carbohydrate determinants (CCDs). RESULTS: Sensitization to Cor a 14 was observed in 18 out of 20 preschool children, 8 out of 10 school-aged children and 2 out of 7 adults with generalized reactions and in 3 out of 14 infants with AD. Only 2 out of 38 patients with an oral allergy syndrome (OAS) were sensitized to Cor a 14. No sensitization to Cor a 14 was observed in the group of hazelnut-tolerant and healthy control individuals. Sensitization to Cor a 1.04 was seen in 36 out of 38 OAS patients and in 14 out of 37 patients with generalized reactions. However, only 3 patients with generalized reactions were monosensitized to Cor a 1.04. Sensitization to Cor a 9 was observed in 26 out of 37 patients with generalized reactions and in 4 out of 14 infants with AD. Sensitization to Cor a 11, Cor a 8, rBet v 2 and CCDs was rare. CONCLUSIONS: Sensitization to Cor a 14 can have early onset and shows age-related variations. Together with Cor a 9, Cor a 14 enables us to correctly identify almost 90% of children with generalized reactions to hazelnut.

Beninese Plant Extracts with Antiplasmodial Activity Select New Allele Variants Msp1 and Msp2 in Plasmodium falciparum.

Background: Natural medicinal products are commonly used as a remedy against malaria infections in African populations and have become a major source of information for the screening of new and more effective antiplasmodial molecules. Therefore, in vitro studies are needed to validate the efficacy of these medicinal products and to explore the potential effects of such drugs on the genetic diversity of Plasmodium falciparum. The current study has investigated the impact of some Beninese plant extracts with antiplasmodial activity on the genetic diversity of P. falciparum. Method: Five (5) ethanolic plant extracts (Dissotis rotundifolia, Ehretia cymosa Thonn, Hibiscus surattensis L., Cola millenii K. Shum, and Costus afer Ker Gawl) and a compound extracted from Ehretia cymosa Thonn (encoded CpE2) were tested against asexual stage parasites of a culture-adapted strain of P. falciparum. Subsequently, the P. falciparum Msp1 and Msp2 markers were genotyped, and the number of allelic variants and the multiplicity of infection (MOI) were compared between drug-exposed and unexposed parasites. Results: All plant extracts have shown inhibitory activity against asexual P. falciparum and selected new allelic variants of the Msp1 and Msp2 genes compared to unexposed parasites. The newly selected allelic variants were K1_100bp and RO33_300bp of the Msp1 gene and FC27_150bp, FC27_300bp, FC27_400bp, and FC27_600bp of the Msp2 gene. However, there was no significant difference in MOI between drug-exposed and unexposed parasites. Conclusion: Our study highlights a source for the selection of new Msp1 and Msp2 alleles after exposure to antimalarial drugs. These findings pave the way for further studies investigating the true roles of these newly selected alleles in P. falciparum.

Parallel evolution in mosquito vectors - a duplicated esterase locus is associated with resistance to pirimiphos-methyl in An. gambiae.

The primary control methods for the African malaria mosquito, Anopheles gambiae, are based on insecticidal interventions. Emerging resistance to these compounds is therefore of major concern to malaria control programmes. The organophosphate, pirimiphos-methyl, is a relatively new chemical in the vector control armoury but is now widely used in indoor residual spray campaigns. Whilst generally effective, phenotypic resistance has developed in some areas in malaria vectors. Here, we used a population genomic approach to identify novel mechanisms of resistance to pirimiphos-methyl in Anopheles gambiae s.l mosquitoes. In multiple populations, we found large and repeated signals of selection at a locus containing a cluster of detoxification enzymes, some of whose orthologs are known to confer resistance to organophosphates in Culex pipiens. Close examination revealed a pair of alpha-esterases, Coeae1f and Coeae2f, and a complex and diverse pattern of haplotypes under selection in An. gambiae, An. coluzzii and An. arabiensis. As in Cx. pipiens, copy number variation seems to play a role in the evolution of insecticide resistance at this locus. We used diplotype clustering to examine whether these signals arise from parallel evolution or adaptive introgression. Using whole-genome sequenced phenotyped samples, we found that in West Africa, a copy number variant in Anopheles gambiae is associated with resistance to pirimiphos-methyl. Overall, we demonstrate a striking example of contemporary parallel evolution which has important implications for malaria control programmes.

Asymptomatic Plasmodium infection among primary schoolchildren and Anopheles-mediated malaria transmission: A cross-sectional study in Ouidah; south-western Benin.

Understanding the contribution of asymptomatic Plasmodium carriers in malaria transmission might be helpful to design and implement new control measures. The present study explored the prevalence of asymptomatic and symptomatic Plasmodium infections (asexual and sexual stages) and the contribution of asymptomatic P. falciparum carriers to Anopheles-mediated malaria transmission in Ouidah (Benin). Thick and thin blood smears were examined from finger-prick blood specimens using light microscopy, and the density of both asexual and sexual stages of Plasmodium species was calculated. Infectivity of gametocyte-infected blood samples to Anopheles gambiae was assessed through direct membrane feeding assays. The prevalence of asymptomatic Plasmodium infections was 28.73% (289/1006). All the asymptomatic gametocyte-carriers (19/19), with gametocytaemia ranging from 10 - 1200 gametocytes/µL of blood, were infectious to An. gambiae mosquitoes. The mean oocyst prevalences varied significantly (χ 2  = 16.42, df = 7, p = 0.02) among laboratory mosquito strains (6.9 - 39.4%) and near-field mosquitoes (4.9 - 27.2%). Likewise, significant variation (χ 2  = 56.85, df = 7, p = 6.39 × 10-10) was observed in oocyst intensity. Our findings indicate that asymptomatic Plasmodium carriers could significantly contribute to malaria transmission. Overall, this study highlights the importance of diagnosing and treating asymptomatic and symptomatic infection carriers during malaria control programmes.