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

Pharmacokinetics, safety, and efficacy of a single co-administered dose of diethylcarbamazine, albendazole and ivermectin in adults with and without Wuchereria bancrofti infection in Cote d\u27Ivoire

BackgroundA single co-administered dose of ivermectin (IVM) plus diethylcarbamazine (DEC) plus albendazole (ALB), or triple-drug therapy, was recently found to be more effective for clearing microfilariae (Mf) than standard DEC plus ALB currently used for mass drug administration programs for lymphatic filariasis (LF) outside of sub-Saharan Africa. Triple-drug therapy has not been previously tested in LF-uninfected individuals from Africa. This study evaluated the pharmacokinetics (PK), safety, and efficacy of triple-drug therapy in people with and without Wuchereria bancrofti infection in West Africa.MethodsIn this open-label cohort study, treatment-naïve microfilaremic (>50 mf/mL, n = 32) and uninfected (circulating filarial antigen negative, n = 24) adults residing in Agboville district, Côte d’Ivoire, were treated with a single dose of IVM plus DEC plus ALB, and evaluated for adverse events (AEs) until 7 days post treatment. Drug levels were assessed by liquid chromatography and mass spectrometry. Persons responsible for assessing AEs were blinded to participants’ infection status.FindingsThere was no difference in AUC0-inf or Cmax between LF-infected and uninfected participants (P>0.05 for all comparisons). All subjects experienced mild AEs; 28% and 25% of infected and uninfected participants experienced grade 2 AEs, respectively. There were no severe or serious adverse events. Only fever (16 of 32 versus 4 of 24, PConclusionsModerate to heavy W. bancrofti infection did not affect PK parameters for IVM, DEC or ALB following a single co-administered dose of these drugs compared to uninfected individuals. The drugs were well tolerated. This study confirmed the efficacy of the triple-drug therapy for clearing W. bancrofti Mf and has added important information to support the use of this regimen in LF elimination programs in areas of Africa without co-endemic onchocerciasis or loiasis.Trial registrationClinicalTrials.gov NCT02845713.</div

Malaria knowledge and long-lasting insecticidal net use in rural communities of central Côte d'Ivoire

ABSTRACT: BACKGROUND: To improve effectiveness of malaria control interventions, it is essential to deepen the knowledge of contextual factors that govern people's practice for preventive and curative measures. The aim of this study was to determine factors that influence the use of long-lasting insecticidal nets (LLINs) in three rural communities of Cote d'Ivoire, two of which benefited from recent interventions. METHODS: The study was carried out in 957 households in three villages (Bozi, N'Dakonankro and Yoho) located in central Cote d'Ivoire. Indicators of socioeconomic position (SEP), malaria knowledge and practice, placing special emphasis on LLINs, were investigated during a cross-sectional questionnaire survey. Principal component analysis was used to calculate the SEP of households by means of a list of household assets ownership. The concentration index was used to assess the direction of the association between SEP and a given variable. To compare groups or means, Fisher's exact test, chi2 and Kruskal-Wallis test were used, as appropriate. RESULTS: Significant differences were found between SEP and reported malaria symptoms, such as fever or hot body, convulsion, anaemia and jaundice (yellow eyes). Individuals from the least poor group cited more often the use of bed nets and insecticide-treated nets (ITNs) compared to poorer groups. The mean number of individuals reporting the use of bed nets and LLINs was different between groups with different educational level. Moreover, the mean number of LLINs in a household was influenced by the presence of children below five years of age. CONCLUSION: The study not only confirmed that education and SEP play important roles in the prevention and control of malaria and promotion of health in general, but pointed at the basic essential knowledge and the key behavioural elements that should guide education and learning processes among the poorer segments of the population. In turn, such knowledge may change behaviour and lead to an increased utilization of LLIN

Association of Reduced Long-Lasting Insecticidal Net Efficacy and Pyrethroid Insecticide Resistance With Overexpression of CYP6P4, CYP6P3, and CYP6Z1 in Populations of Anopheles coluzzii From Southeast Côte d'Ivoire.

BACKGROUND: Resistance to major public health insecticides in Côte d'Ivoire has intensified and now threatens the long-term effectiveness of malaria vector control interventions. METHODS: This study evaluated the bioefficacy of conventional and next-generation long-lasting insecticidal nets (LLINs), determined resistance profiles, and characterized molecular and metabolic mechanisms in wild Anopheles coluzzii from Southeast Côte d'Ivoire in 2019. RESULTS: Phenotypic resistance was intense: >25% of mosquitoes survived exposure to 10 times the doses of pyrethroids required to kill susceptible populations. Similarly, the 24-hour mortality rate with deltamethrin-only LLINs was very low and not significantly different from that with an untreated net. Sublethal pyrethroid exposure did not induce significant delayed vector mortality effects 72 hours later. In contrast, LLINs containing the synergist piperonyl butoxide, or new insecticides clothianidin and chlorfenapyr, were highly toxic to A. coluzzii. Pyrethroid-susceptible A. coluzzii were significantly more likely to be infected with malaria, compared with those that survived insecticidal exposure. Pyrethroid resistance was associated with significant overexpression of CYP6P4, CYP6P3, and CYP6Z1. CONCLUSIONS: Study findings raise concerns regarding the operational failure of standard LLINs and support the urgent deployment of vector control interventions incorporating piperonyl butoxide, chlorfenapyr, or clothianidin in areas of high resistance intensity in Côte d'Ivoire

Reduced long-lasting insecticidal net efficacy and pyrethroid insecticide resistance are associated with over-expression of <i>CYP6P4, CYP6P3</i> and <i>CYP6Z1</i> in populations of <i>Anopheles coluzzii</i> from South-East Côte d’Ivoire

AbstractBackgroundResistance to major public health insecticides in Côte d’Ivoire has intensified and now threatens the long-term effectiveness of malaria vector control interventions.MethodsThis study evaluated the bioefficacy of conventional and next-generation long-lasting insecticidal nets (LLINs), determined resistance profiles, and characterized molecular and metabolic mechanisms in wild Anopheles coluzzii from South-East Côte d’Ivoire in 2019.ResultsPhenotypic resistance was intense: more than 25% of mosquitoes survived exposure to ten times the doses of pyrethroids required to kill susceptible populations. Similarly, 24-hour mortality to deltamethrin-only LLINs was very low and not significantly different to an untreated net. Sub-lethal pyrethroid exposure did not induce significant delayed vector mortality 72 hours later. In contrast, LLINs containing the synergist piperonyl butoxide (PBO), or new insecticides, clothianidin and chlorfenapyr, were highly toxic to An. coluzzii. Pyrethroid-susceptible An. coluzzii were significantly more likely to be infected with malaria, compared to those that survived insecticidal exposure. Pyrethroid resistance was associated with significant over-expression of CYP6P4, CPY6Z1 and CYP6P3.ConclusionsStudy findings raise concerns regarding the operational failure of standard LLINs and support the urgent deployment of vector control interventions incorporating PBO, chlorfenapyr or clothianidin in areas of high resistance intensity in Côte d’Ivoire.</jats:sec

Joint spatial modelling of malaria incidence and vector's abundance shows heterogeneity in malaria‐vector geographical relationships

Limited attention from the modelling community has been given to ecological approaches which aim to predict geographical patterns of malaria by accounting for the joint effects of different vectors and environmental drivers. A hierarchical multivariate joint spatial Gaussian generalised linear model was developed to provide joint parameters inference and mapping of counts of Anopheles gambiae, An. funestus, An. nili and malaria incidence collected in an area of Cote d'Ivoire. Variable‐selection methods were applied to select important predictors for each mosquito species and malaria incidence. The proposed joint model led to a general reduction of the variance in the estimates compared to independent modelling. There was high variability in the composition of Anopheles mosquito species in the villages with each species suitability only partly overlapping geographically. Abundances of An. gambiae, An. funestus and An. nili were primarily determined by temperature. None of the species were found as a significant predictor for the others. Anopheles gambiae was the predominant species and only An. gambiae female abundance was an important variable (linear predictor) for malaria incidence. However, the geographic correlation analyses show that the rest of Anopheles species are likely playing a role in malaria suitability. Residuals from the models of mosquito abundance and malaria cases are also correlated with each other and overlapping but in geographic patches, meaning that local drivers of vector‐malaria suitability are still present and not represented by the predictors used in the model. Synthesis and applications: Joint modelling improve predictive estimation compared to individual modelling. The accurate predictions highlighted high diversity in the association between malaria and vector species, with most of the area having more than one species suitability correlated with malaria suitability. These zones are unlikely to benefit from species‐specific interventions. Areas with correlated malaria and vector species suitability residuals contain local information, not included in the model, that requires further investigation. This will identify additional communal malaria and vectors factors that need to be considered for optimal malaria control and elimination strategies since these factors are expected to be linked to the local malaria transmission

Education and Socio‑economic status are key factors influencing use of insecticides and malaria knowledge in rural farmers in Southern Côte d’Ivoire

Background Insecticides play a key role in rural farming; however, their over- or misuse has been linked with a negative impact on malaria vector control policies. This study was conducted amongst agricultural communities in Southern Côte d’Ivoire to identify which insecticides are used by local farmers and how it relates to the perception of farmers on malaria. Understanding the use of insecticides may help in designing awareness programme on mosquito control and pesticides management. Methods A questionnaire was administered to 1399 farming households across ten villages. Farmers were interviewed on their education, farming practices (e.g. crops cultivated, insecticides use), perception of malaria, and the different domestic strategies of mosquito control they use. Based on some pre-defined household assets, the socioeconomic status (SES) of each household was estimated. Statistical associations were calculated between different variables, showing significant risk factors. Results The educational level of farmers was significantly associated with their SES (p < 0.0001). Most of the householders (88.82%) identified mosquitoes as the principal cause of malaria, with good knowledge of malaria resulting as positively related to high educational level (OR = 2.04; 95%CI: 1.35, 3.10). The use of indoor chemical compounds was strongly associated to the SES of the households, their education level, their use of ITNs and insecticide in agricultural (p < 0.0001). Indoor application of pyrethroid insecticides was found to be widespread among farmers as well as the use of such insecticide for crops protection. Conclusion Our study shows that the education level remains the key factor influencing the use of insecticides by farmers and their awareness of malaria control. We suggest that better communication tailored to education level and including SES, controlled availability and access to chemical products, should be considered when designing campaigns on use of pesticides and vector borne disease control for local communities

Widespread occurrence of copy number variants and fixation of pyrethroid target site resistance in Anopheles gambiae (s.l.) from southern Cote d’Ivoire

Resistance to pyrethroid and organophosphate insecticides in the malaria vector Anopheles gambiae (s.l.) is conferred by a variety of genetic mutations, including single nucleotide polymorphisms (SNPs) and copy number variants (CNVs). Knowledge of the distribution of these mutations in mosquito populations is a prerequisite for establishing better strategies for their management. In this study, a total of 755 Anopheles gambiae (s.l.) from southern Côte d’Ivoire were exposed to deltamethrin or pirimiphos-methyl insecticides and were screened to assess the distribution of SNPs and CNVs known or believed to confer resistance to one or other of the insecticide classes. Most individuals from the An. gambiae (s.l.) complex were identified by molecular tests as Anopheles coluzzii. Survival to deltamethrin (from 94% to 97%) was higher than to pirimiphos-methyl (from 10% to 49%). In An. gambiae (s.s.), the SNP in the Voltage Gated Sodium Channel (Vgsc) at the 995F locus (Vgsc-995F) was fixed, while other target site mutations were rare or absent (Vgsc-402L: 0%; Vgsc-1570Y: 0%, Acetylcholinesterase Acel-280S: 14%). In An. coluzzii, Vgsc-995F was the target site SNP found at highest frequency (65%) followed by other target site mutations (Vgsc-402L: 36%; Vgsc-1570Y: 0.33%; Acel-280S: 45%). The Vgsc-995S SNP was not present. The presence of the Ace1-280S SNP was found to be significantly linked to the presence of the Ace1-CNV, Ace1_AgDup. Significant association was found between the presence of the Ace1_AgDup and pirimiphos-methyl resistance in An. gambiae (s.s.) but not in An. coluzzii. The deletion Ace1_Del97 was found in one specimen of An. gambiae (s.s.). Four CNVs in the Cyp6aa/Cyp6p gene cluster, which contains genes of known importance for resistance, were detected in An. coluzzii, the most frequent being Dup 7 (42%) and Dup 14 (26%). While none of these individual CNV alleles were significantly associated with resistance, copy number in the Cyp6aa gene region in general was associated with increased resistance to deltamethrin. Elevated expression of Cyp6p3 was nearly associated with deltamethrin resistance, although there was no association of resistance with copy number. Use of alternative insecticides and control methods to arrest resistance spread in An. coluzzii populations is merited

Entomological impact of three years of clothianidin-based indoor residual spraying in two high malaria endemic districts in Côte d’Ivoire

Background: Indoor residual spraying (IRS) represents a major malaria vector control strategy to reduce malaria burden. The first major IRS campaign for malaria control in Côte d’Ivoire was implemented by the US Presidents Malaria Initiative in two malaria endemic districts between 2020 and 2022. This study report on the effect of clothianidin-based IRS on key entomological indices of malaria transmission in the IRS districts following three rounds of IRS with clothianidin-based insecticide formulations. Methods: Pre- and post-IRS adult mosquito collections were conducted monthly in two IRS districts (Sakassou and Nassian) and two control districts (Beoumi and Dabakala) using human landing catches (HLCs) and pyrethrum spray catches (PSC) from April 2019 through December 2022. Mosquitoes collected were morphologically identified, further analysed for sibling species using PCR, and tested for sporozoite infection using the enzyme-linked immunosorbent assay (ELISA). A proportion of the Anopheles gambiae sensu lato mosquitoes collected from HLCs were also dissected to assess their parity rates. Results: Anopheles coluzzii and An. gambiae were the main malaria vectors collected in the study area. In the IRS arm HBR declined progressively across spray rounds, with a statistically significant reduction of about 60% by the third round (IRR = 0.40; p = 0.002) but not in the controls (all p &gt; 0.5). The odds of sporozoite infection decreased by 49% after the first (OR = 0.51; p = 0.023) and second (OR = 0.51, p = 0.043) at the IRS sites but did not show any further decline by the third round (p = 0.51). These reductions resulted in four-fold decrease in EIR from 3.92 infective/bites/person/night pre-IRS to 1.25 ib/p/n post-IRS. EIR in control declined only marginally from 0.91ib/p/n pre-IRS to 0.58 ib/p/n post-IRS. Conclusions: Clothianidin-based IRS was associated with a clear reduction in An. gambiae and An. coluzzii biting densities in the IRS districts and, more importantly, a significant reduction in EIR after accounting for changes over the same period in the neighbouring control areas. These findings are consistent with the epidemiological findings reported in the same sites and demonstrates the utility of IRS as an important tool for malaria control in a highly endemic setting like Côte d’Ivoire.</p

Assessing species composition and insecticide resistance of Anopheles gambiae complex members in three coastal health districts of Côte d’Ivoire

Although malaria is endemic in coastal Côte d’Ivoire, updated data on the resistance profile of the main vector, Anopheles gambiae sensu lato (s.l.), are still lacking, thus compromising decision-making for an effective vector control intervention. This study investigated the complex members and the insecticide resistance in the Anopheles gambiae s.l. populations in coastal Côte d’Ivoire. Between 2018 and 2020, cross sectional survey bioassays were conducted on female An. gambiae s.l. mosquitoes in three coastal health districts (Aboisso, Jacqueville and San Pedro) of Côte d’Ivoire. Pyrethroids deltamethrin, permethrin and alphacypermethrin (1X, 5X and 10X), clothianidin and synergist piperonyl butoxide (PBO) combined with pyrethroid 1X were tested using WHO tube bioassays. Chlorfenapyr was evaluated using CDC bottle bioassays. An. gambiae complex members and kdr 995F, kdr 995S and Ace-1 280S mutations were identified using polymerase chain reaction (PCR) technique. Overall, An. gambiae s.l. populations were primarily composed of Anopheles coluzzii (88.24%, n = 312), followed by Anopheles gambiae sensu stricto (7.56%) and hybrids (4.17%). These populations displayed strong resistance to pyrethroids at standard diagnostic doses, with mortality remaining below 98% even at 10X doses, except for alphacypermethrin in Aboisso. Pre-exposure to PBO significantly increased mortality but did not induce susceptibility, except for alphacypermethrin in Jacqueville. Clothianidin induced full susceptibility in Jacqueville and San Pedro, while chlorfenapyr induced susceptibility in Aboisso at 100 μg ai/bottle and all three districts at 200 μg ai/bottle. kdr 995F mutation dominated, with frequencies varying from 71.2% to 79.3%. kdr 995S had low, rates with frequencies ranging from 2.3% to 5.7%. Ace-1 280S prevalence varied between 4.2% and 42.9%. Coastal Côte d’Ivoire’s An. gambiae s.l. populations were mainly composed of An. coluzzii and showed high resistance to pyrethroids. Clothianidin, chlorfenapyr, and PBO with pyrethroids increased mortality, indicating their potential use as an alternative for malaria vector control