Modelling spatiotemporal trends in the frequency of genetic mutations conferring insecticide target-site resistance in African mosquito malaria vector species.

BACKGROUND: Resistance in malaria vectors to pyrethroids, the most widely used class of insecticides for malaria vector control, threatens the continued efficacy of vector control tools. Target-site resistance is an important genetic resistance mechanism caused by mutations in the voltage-gated sodium channel (Vgsc) gene that encodes the pyrethroid target-site. Understanding the geographic distribution of target-site resistance, and temporal trends across different vector species, can inform strategic deployment of vector control tools. RESULTS: We develop a Bayesian statistical spatiotemporal model to interpret species-specific trends in the frequency of the most common resistance mutations, Vgsc-995S and Vgsc-995F, in three major malaria vector species Anopheles gambiae, An. coluzzii, and An. arabiensis over the period 2005-2017. The models are informed by 2418 observations of the frequency of each mutation in field sampled mosquitoes collected from 27 countries spanning western and eastern regions of Africa. For nine selected countries, we develop annual predictive maps which reveal geographically structured patterns of spread of each mutation at regional and continental scales. The results show associations, as well as stark differences, in spread dynamics of the two mutations across the three vector species. The coverage of ITNs was an influential predictor of Vgsc allele frequencies, with modelled relationships between ITN coverage and allele frequencies varying across species and geographic regions. We found that our mapped Vgsc allele frequencies are a significant partial predictor of phenotypic resistance to the pyrethroid deltamethrin in An. gambiae complex populations. CONCLUSIONS: Our predictive maps show how spatiotemporal trends in insecticide target-site resistance mechanisms in African An. gambiae vary across individual vector species and geographic regions. Molecular surveillance of resistance mechanisms will help to predict resistance phenotypes and track their spread.

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for antimalarial intervention deployment.

Studies of insecticide resistance provide insights into the capacity of populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs). Resistance in the major malaria vectors has increased in concert with the expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance is crucial for the development and deployment of resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236 M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolize pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets cotreated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid cotreated LLINs across African countries.

Determination of the discriminating concentration of chlorfenapyr (pyrrole) and Anopheles gambiae sensu lato susceptibility testing in preparation for distribution of Interceptor® G2 insecticide-treated nets.

BACKGROUND: Following agricultural use and large-scale distribution of insecticide-treated nets (ITNs), malaria vector resistance to pyrethroids is widespread in sub-Saharan Africa. Interceptor® G2 is a new dual active ingredient (AI) ITN treated with alpha-cypermethrin and chlorfenapyr for the control of pyrethroid-resistant malaria vectors. In anticipation of these new nets being more widely distributed, testing was conducted to develop a chlorfenapyr susceptibility bioassay protocol and gather susceptibility information. METHODS: Bottle bioassay tests were conducted using five concentrations of chlorfenapyr at 12.5, 25, 50, 100, and 200 µg AI/bottle in 10 countries in sub-Saharan Africa using 13,639 wild-collected Anopheles gambiae sensu lato (s.l.) (56 vector populations per dose) and 4,494 pyrethroid-susceptible insectary mosquitoes from 8 colonized strains. In parallel, susceptibility tests were conducted using a provisional discriminating concentration of 100 µg AI/bottle in 16 countries using 23,422 wild-collected, pyrethroid-resistant An. gambiae s.l. (259 vector populations). Exposure time was 60 min, with mortality recorded at 24, 48 and 72 h after exposure. RESULTS: Median mortality rates (up to 72 h after exposure) of insectary colony mosquitoes was 100% at all five concentrations tested, but the lowest dose to kill all mosquitoes tested was 50 µg AI/bottle. The median 72-h mortality of wild An. gambiae s.l. in 10 countries was 71.5, 90.5, 96.5, 100, and 100% at concentrations of 12.5, 25, 50, 100, and 200 µg AI/bottle, respectively. Log-probit analysis of the five concentrations tested determined that the LC95 of wild An. gambiae s.l. was 67.9 µg AI/bottle (95% CI: 48.8-119.5). The discriminating concentration of 203.8 µg AI/bottle (95% CI: 146-359) was calculated by multiplying the LC95 by three. However, the difference in mortality between 100 and 200 µg AI/bottle was minimal and large-scale testing using 100 µg AI/bottle with wild An. gambiae s.l. in 16 countries showed that this concentration was generally suitable, with a median mortality rate of 100% at 72 h. CONCLUSIONS: This study determined that 100 or 200 µg AI/bottle chlorfenapyr in bottle bioassays are suitable discriminating concentrations for monitoring susceptibility of wild An. gambiae s.l., using mortality recorded up to 72 h. Testing in 16 countries in sub-Saharan Africa demonstrated vector susceptibility to chlorfenapyr, including mosquitoes with multiple resistance mechanisms to pyrethroids.

Intensity of pyrethroid resistance in Anopheles gambiae before and after a mass distribution of insecticide-treated nets in Kinshasa and in 11 provinces of the Democratic Republic of Congo.

BACKGROUND: Between 2011 and 2018, an estimated 134.8 million pyrethroid-treated long-lasting insecticidal nets (LLINs) were distributed nationwide in the Democratic Republic of Congo (DRC) for malaria control. Pyrethroid resistance has developed in DRC in recent years, but the intensity of resistance and impact on LLIN efficacy was not known. Therefore, the intensity of resistance of Anopheles gambiae sensu lato (s.l.) to permethrin and deltamethrin was monitored before and after a mass distribution of LLINs in Kinshasa in December 2016, and in 6 other sites across the country in 2017 and 11 sites in 2018. METHODS: In Kinshasa, CDC bottle bioassays using 1, 2, 5, and 10 times the diagnostic dose of permethrin and deltamethrin were conducted using An. gambiae s.l. collected as larvae and reared to adults. Bioassays were conducted in four sites in Kinshasa province 6 months before a mass distribution of deltamethrin-treated LLINs and then two, six, and 10 months after the distribution. One site in neighbouring Kongo Central province was used as a control (no mass campaign of LLIN distribution during the study). Nationwide intensity assays were conducted in six sites in 2017 using CDC bottle bioassays and in 11 sites in 2018 using WHO intensity assays. A sub-sample of An. gambiae s.l. was tested by PCR to determine species composition and frequency of kdr-1014F and 1014S alleles. RESULTS: In June 2016, before LLIN distribution, permethrin resistance intensity was high in Kinshasa; the mean mortality rate was 43% at the 5× concentration and 73% at the 10× concentration. Bioassays at 3 time points after LLIN distribution showed considerable variation by site and time and there was no consistent evidence for an increase in pyrethroid resistance intensity compared to the neighbouring control site. Tests of An. gambiae s.l. in 6 sites across the country in 2017 and 11 sites in 2018 showed all populations were resistant to the diagnostic doses of 3 pyrethroids. In 2018, the intensity of resistance varied by site, but was generally moderate for all three pyrethroids, with survivors at ×5 the diagnostic dose. Anopheles gambiae sensu stricto (s.s.) was the most common species identified across 11 sites in DRC, but in Kinshasa, An. gambiae s.s. (91%) and Anopheles coluzzii (8%) were sympatric. CONCLUSIONS: Moderate or high intensity pyrethroid resistance was detected nationwide in DRC and is a serious threat to sustained malaria control with pyrethroid LLINs. Next generation nets (PBO nets or bi-treated nets) should be considered for mass distribution.

The current distribution and characterization of the L1014F resistance allele of the kdr gene in three malaria vectors (Anopheles gambiae, Anopheles coluzzii, Anopheles arabiensis) in Benin (West Africa).

BACKGROUND: The fight against malaria faces various biological obstacles, including the resistance of parasites to anti-malarial drugs and the resistance of mosquito vectors to insecticides. The resistance of Anopheles gambiae sensu lato (s.l.) to pyrethroids, the only class of insecticides used to impregnate mosquito nets, is known in Benin; the expansion of this resistance is influenced by the existence of gene flow between species, otherwise by the presence or absence of the kdr mutation in them. The objective of this study is to determine the spatial distribution of An. gambiae and the level of expression of the pyrethroid resistance kdr gene in seven agro-ecological zones of Benin. METHODS: The study was conducted in 18 localities belonging to seven agro-ecological zones where environmental parameters varied. The sites represent the main areas of eco-epidemiological malaria in Benin. Anopheles gambiae larvae were collected in natural breeding sites using ladles and dipping method and reared under standard conditions. These larvae were reared under standard conditions of temperature and humidity (26 to 30 °C and 60 to 90%) at the insectarium of the Centre de Recherche Entomologique de Cotonou (CREC). Adult female mosquitoes having emerged are morphologically and molecularly identified. Homozygous resistant (1014F/1014F), homozygous sensitive (1014L/1014L) and heterozygous (1014F/1014L) genotypes of the L1014F kdr gene mutation are determined by PCR. RESULTS: A total of 677 An. gambiae was subjected at the PCR. The results revealed the presence of three vector species of the An. gambiae complex, of which 409 Anopheles coluzzii, 259 An. gambiae, 5 hybrids (An. coluzzii/An. gambiae) and 4 Anopheles arabiensis in the different agro-ecological zones. The four An. arabiensis were only found in Dassa, a locality in the cotton zone of central Benin. The frequency of distribution of the L1014F allele of the kdr gene varies from 84.48 to 100% in An. gambiae, from 80 to 100% in An. coluzzii and from 0 to 75% in An. arabiensis in the different agro-ecological zones. Moreover, a significant difference is generally observed in the distribution of the L1014F allele (P < 0.05). By comparing in pairs the distribution frequencies of this allele in the two species by agro-ecological zone, only a significant difference is noted in the central cotton and fishery zones (P = 0.0496). CONCLUSION: In summary, even if the data are in small portions, the An. Arabiensis species was found only in central Benin and the L1014F allele of the kdr gene is widespread and seems to fix in all the species recorded in the different agro-ecological zones. This situation amplifies the problem of resistance, which could eventually be a significant obstacle for the malaria vectors control. Similarly, a study of their genetic structure via the L1014F allele is necessary in order to put in place strategies to manage this resistance. These strategies will take into account both the ecology and the genetic diversity of the organisms involved to preserve the effectiveness of pyrethroids, the only insecticides used for the impregnation of mosquito nets.

Pesticides and the evolution of the genetic structure of Anopheles coluzzii populations in some localities in Benin (West Africa).

BACKGROUND: Changes in the natural habitats of insect groups are determined the genetic polymorphisms between individuals. The objective of this study was to establish the genetic structure of the Anopheles coluzzii populations in four localities of Benin. METHODS: Insecticide surveys and larval sampling were conducted on 4 study localities, including Cotonou, Ketou, Zagnanado, and Sô-Ava. Molecular characterizations were performed on the Anopheles mosquitoes collected with the allelic and genotypic frequencies of kdr gene determined. The multiple comparison Chi square test for proportions was performed with R version 3.3.3. Next, the observed heterozygosity, expected heterozygosity, and indices of fixation, and genetic differentiation were estimated. Finally, the Hardy-Weinberg equilibrium (EHW) was determined to assess whether panmixia exists in the different populations of mosquitoes of the agroecological zones under study. RESULTS: Carbamates, pyrethroids, organophosphorus and organochlorines use have been reported in all localities except Sô-Ava. Anopheles coluzzii was strongly represented across all study localities. The L1014F allele was observed in the localities of Kétou, Cotonou and Zagnanado. Likewise, insecticide selection pressure of homozygous resistant individuals (L1014F/L1014F) was significantly higher in Kétou, Cotonou and Zagnanado (p value < 0.05). Surprisingly in Sô-Ava, a relatively high frequency of the L1014F allele despite the reported absence of pesticide use was observed. All mosquito populations were found to be deficient in heterozygosity across the study sites (FIS< 0). No genetic differentiation (FST< 0) was observed in the localities of Zagnanado and Kétou. CONCLUSION: The survey on the use of insecticides showed that insecticide selection pressures differ across the investigated localities. It would be desirable to rotate or apply formulations of combined products with different modes of action. Doing so would enable a better management of resistant homozygous individuals, and mitigate the resistance effect of commonly used insecticides.

Susceptibility testing of Anopheles malaria vectors with the neonicotinoid insecticide clothianidin; results from 16 African countries, in preparation for indoor residual spraying with new insecticide formulations.

BACKGROUND: In 2017, more than 5 million house structures were sprayed through the U.S. President's Malaria Initiative, protecting more than 21 million people in sub-Saharan Africa. New IRS formulations, SumiShield™ 50WG and Fludora Fusion™ WP-SB, became World Health Organization (WHO) prequalified vector control products in 2017 and 2018, respectively. Both formulations contain the neonicotinoid active ingredient, clothianidin. The target site of neonicotinoids represents a novel mode of action for vector control, meaning that cross-resistance through existing mechanisms is less likely. In preparation for rollout of clothianidin formulations as part of national IRS rotation strategies, baseline susceptibility testing was conducted in 16 countries in sub-Saharan Africa. METHODS: While work coordinated by the WHO is ongoing to develop a suitable bottle bioassay procedure, there was no published guidance regarding clothianidin susceptibility procedures or diagnostic concentrations. Therefore, a protocol was developed for impregnating filter papers with 2% w/v SumiShield™ 50WG dissolved in distilled water. Susceptibility tests were conducted using insectary-reared reference Anopheles and wild collected malaria vector species. All tests were conducted within 24 h of treating papers, with mortality recorded daily for 7 days, due to the slow-acting nature of clothianidin against mosquitoes. Anopheles gambiae sensu lato (s.l.) adults from wild collected larvae were tested in 14 countries, with wild collected F0 Anopheles funestus s.l. tested in Mozambique and Zambia. RESULTS: One-hundred percent mortality was reached with all susceptible insectary strains and with wild An. gambiae s.l. from all sites in 11 countries. However, tests in at least one location from 5 countries produced mortality below 98%. While this could potentially be a sign of clothianidin resistance, it is more likely that the diagnostic dose or protocol requires further optimization. Repeat testing in 3 sites in Ghana and Zambia, where possible resistance was detected, subsequently produced 100% mortality. Results showed susceptibility to clothianidin in 38 of the 43 sites in sub-Saharan Africa, including malaria vectors with multiple resistance mechanisms to pyrethroids, carbamates and organophosphates. CONCLUSIONS: This study provides an interim diagnostic dose of 2% w/v clothianidin on filter papers which can be utilized by National Malaria Control Programmes and research organizations until the WHO concludes multi-centre studies and provides further guidance.

Small-scale field evaluation of the efficacy and residual effect of Fludora® Fusion (mixture of clothianidin and deltamethrin) against susceptible and resistant Anopheles gambiae populations from Benin, West Africa.

BACKGROUND: In recognition of the threat of insecticide resistance in vectors of malaria, the WHO Global Malaria Programme recommends the development of an appropriate and comprehensive response to insecticide resistance. In principle, good resistance management practice requires the application of multiple insecticides of different modes of action, for example, in rotations and mixtures. Insecticides recommended by the World Health Organization for indoor residual spraying and long-lasting insecticide nets are limited. It is, therefore, judicious to prevent the rapid spread of insecticide resistance by evaluating new insecticides formulations with different modes of action and long residual effect. METHODS: Fludora® Fusion, a new neonicotinoid IRS formulation (a mixture of 500 g/kg clothianidin and 62.5 g/kg deltamethrin applied 200 mg ai/sqm + 25 mg ai/sqm, respectively) was tested. Small scale field evaluation of this product was conducted in the district of Dangbo in Benin, to compare its efficacy and residual effect on cement and mud walls against those of clothianidin 200 mg ai/sqm (WG 70) alone, and of deltamethrin 25 mg ai/sqm (WG 250) alone. WHO wall cone bioassays were conducted monthly with laboratory susceptible Anopheles "Kisumu" and wild Anopheles gambiae sensu stricto (s.s.) population from Dangbo. The induced mortality by each treatment per wall substrate for 24 h, 48 h, and 72 h post exposure were recorded every month and analysed. RESULTS: Fludora® Fusion and clothianidin WG 70 showed mortality rates over 80% WHO bio-efficacy threshold on cement walls either with susceptible or resistant An. gambiae s.s. over a period of 10 and 9 months, respectively. Treatment with Fludora® Fusion and clothianidin WG 70 on the mud walls showed residual effect for 6 months and 5 months respectively against both susceptible and resistant mosquitoes. During the whole evaluation period, deltamethrin WG 250 showed mortality rates below 80% against resistant Anopheles population. Furthermore, the knock down rates observed with the Fludora® Fusion combination were significantly higher (p < 5%) than those induced by Clothiandin WG 70. CONCLUSION: Both the Fludora® Fusion combination and clothianidin alone showed very good and lasting efficacy for IRS against resistant Anopheles with some residual benefit provided by the combination. The residual efficacy of the Fludora® Fusion combination evaluated at 10 months shows this product is a good candidate for IRS interventions.

WHO cone bio-assays of classical and new-generation long-lasting insecticidal nets call for innovative insecticides targeting the knock-down resistance mechanism in Benin.

BACKGROUND: To increase the effectiveness of insecticide-treated nets (ITN) in areas of high resistance, new long-lasting insecticidal nets (LLINs) called new-generation nets have been developed. These nets are treated with the piperonyl butoxide (PBO) synergist which inhibit the action of detoxification enzymes. The effectiveness of the new-generation nets has been proven in some studies, but their specific effect on mosquitoes carrying detoxification enzymes and those carrying both detoxification enzymes and the knock-down resistance gene in Benin is not well known. Thus, the objective of this study is to evaluate the efficacy of LLINs treated with PBO on multi-resistant Anopheles gambiae s.l. METHODS: The study occurred in seven cities in Benin, Abomey, Cotonou, Porto-Novo, Zangnanado, Parakou, Malanville and Tanguiéta, and included ten locations selected on a north-south transect. Mosquito larvae were collected from these sites, and adult females from these larvae were exposed to single-pyrethroid-treated nets (LifeNet, PermaNet 2.0, Olyset Net) and bi-treated nets (PermaNet 3.0 and Olyset Plus) based on their level of resistance and using WHO cone tests following WHO guidelines. RESULTS: The different LLINs showed 100% mortality of the susceptible laboratory strain Kisumu and the resistant strain Ace-1R Kisumu. However, with the resistant laboratory strain kdr-Kisumu, mortality was low (16-32%) for all LLINs except PermaNet 3.0 (82.9%). The mortality of local strains carrying only the kdr mechanism varied from 0 to 47% for the single-pyrethroid-treated LLINs and 9 to 86% for bi-treated LLINs. With local strains carrying several mechanisms of resistance (kdr + detoxification enzymes), the observed mortality with different LLINs was also low except for PermaNet 3.0, which induced significantly higher mortality, usually greater than 75% (p < 0.001), with multi-resistant strains. The inhibition of the mortalities induced by the LLINs (11-96%) on multi-resistant field populations was similar to the inhibition observed with the laboratory strain carrying only the knock-down resistance mechanism (kdr-Kisumu) (p > 0.05). CONCLUSION: This study showed that the new-generation LLINs treated with pyrethroids and PBO showed better efficacy compared to conventional LLINs. Although the addition of PBO significantly increased the mortality of mosquitoes, the significant role of the kdr resistance gene in the low efficacy of LLINs calls for LLIN technology innovation that specifically targets this mechanism.

Six years of experience in entomological surveillance of indoor residual spraying against malaria transmission in Benin: lessons learned, challenges and outlooks.

BACKGROUND: From 2008 to 2013, a prevention intervention against malaria based on indoor residual spraying (IRS) was implemented in Benin. From 2008 to 2012, Ficam M(®), a bendiocarb-containing product was used for house spraying, in association with pirimiphos methyl EC (Actellic EC) in 2013. This operation aimed to strengthen the effectiveness of treated nets so as to expedite the achievement of Millennium Development Goals (MDGs): the reduction of morbidity and mortality due to malaria by 75 % from 2000 to 2015. METHODS: Monitoring and evaluation (M&E) was implemented in order to evaluate the impact of IRS intervention on malaria transmission. Anopheles gambiae s.l. populations were sampled by human landing catch. In addition, window exit traps and pyrethrum spray catches were performed to assess exophagic behaviour of Anopheles vectors the main malaria vector in the treated areas. The residual activity of insecticide in the treated walls was also assessed using WHO bioassay test. RESULTS: The purpose of this project was to draw attention to new challenges and future prospects for the success of IRS in Benin. The main strength of the intervention was a large-scale operation in which more than 80 % of the houses were treated due to the strong adhesion of population. In addition, a significant reduction of the EIR in areas under IRS were observed. However, there were many challenges including the high cost of IRS implementation and the identification of suitable areas to implement IRS. This was because of the low and short residual effect of the insecticides recommended for IRS and the management strategy for vector resistance to insecticides. This indicated that challenges are accompanied by suggested solutions. For the cost of IRS to be accessible to states, then local organizations need to be created in partnership with the National Malaria Control Programme (NMCP) in order to ensure relevant planning and implementation of IRS. CONCLUSION: As an anticipatory measure against vector resistance, this paper proposes various methods, such as periodic IRS based on a combination of two or three insecticides of different classes used in rotation every two or three years.

Footprints of positive selection associated with a mutation (N1575Y) in the voltage-gated sodium channel of Anopheles gambiae.

Insecticide resistance is an ideal model to study the emergence and spread of adaptative variants. In the African malaria mosquito, Anopheles gambiae, this is complemented by a strong public health rationale. In this insect, resistance to pyrethroid and DDT insecticides is strongly associated with the mutations L1014F and L1014S within the para voltage-gated sodium channel (VGSC). Across much of West Africa, 1014F frequency approaches fixation. Here, we document the emergence of a mutation, N1575Y, within the linker between domains III-IV of the VGSC. In data extending over 40 kbp of the VGSC 1575Y occurs on only a single long-range haplotype, also bearing 1014F. The 1014F-1575Y haplotype was found in both M and S molecular forms of An. gambiae in West/Central African sample sites separated by up to 2,000 km. In Burkina Faso M form, 1575Y allele frequency rose significantly from 0.053 to 0.172 between 2008 and 2010. Extended haplotype homozygosity analysis of the wild-type 1575N allele showed rapid decay of linkage disequilibrium (LD), in sharp contrast to the extended LD exhibited by 1575Y. A haplotype with long-range LD and high/increasing frequency is a classical sign of strong positive selection acting on a recent mutant. 1575Y occurs ubiquitously on a 1014F haplotypic background, suggesting that the N1575Y mutation compensates for deleterious fitness effects of 1014F and/or confers additional resistance to insecticides. Haplotypic tests of association suggest the latter: The 1014F-1575Y haplotype confers a significant additive benefit above 1014F-1575N for survival to DDT (M form P = 0.03) and permethrin (S form P = 0.003).

Laboratory and field evaluation of the impact of washings on the effectiveness of LifeNet®, Olyset® and PermaNet® 2.0 in two areas, where there is a high level of resistance of Anopheles gambiae to pyrethroids, Benin, West Africa.

BACKGROUND: An investigation carried out in Benin has shown that, in some areas close to rivers where density of mosquitoes is high, long-lasting, insecticidal bed nets (LLINs) are permanently used. In such areas, LLINs are washed every month. Based on this situation, the 20-wash minimum efficacy advised by the manufacturers would be inadequate. The main goal of this study was to evaluate the effectiveness of LifeNet®, Olyset® and Permanet® 2.0 washed several times against Anopheles gambiae sensu stricto (s.s.) populations, which have developed high resistance to pyrethroids. METHODS: Efficacy of LifeNet®, Olyset® and PermaNet® 2.0 washed 30 and 40 times was expressed in terms of blood-feeding inhibition rate, deterrence, induced exophily and mortality rates. This WHOPES phase II evaluation, conducted in experimental huts in Akron (southern Benin) and in Malanville (northern Benin), was accompanied by WHOPES Phase I evaluation. RESULTS: Over 40 successive washes, LifeNet® induced a mortality rate over 80% in phase I. However, beyond 10 washes, Permanet® 2.0 and Olyset induced dramatically reduced mortality rates, respectively 12.5 and 2.5%. With regard to Phase II results, unwashed LifeNet®, LifeNet® and Olyset® washed 30 and 40 times induced a similar exophily rate per study site (at least 58% in Malanville and at least 71% in Akron). Regarding blood feeding inhibition, LifeNet® and Olyset® washed 30 and 40 times significantly reduced wild An. gambiae s.s. blood feeding showing a similar personal protection as unwashed LifeNet®. LifeNet® washed 30 and 40 times induced mortality rates significantly higher than those induced by Olyset® and Permanet® 2.0 (P < 0,05). CONCLUSION: LifeNet®, followed by Olyset®, have shown good efficacy against host-seeking resistant An. gambiae s.s. population in experimental huts in Benin. Lifenet® have shown to be an effective and promising vector control tool to prevent malaria in areas where repeated washings is a common practice in the community.

Transmission patterns of Plasmodium falciparum by Anopheles gambiae in Benin.

BACKGROUND: To better control malaria, the clear and urgent need is for improved data to inform decision makers, but in several African countries, there is a lack of baseline data on vectors and variation in the intensity of malaria transmission. This has resulted in the implementation of vector control efforts that ignore variation in vector behaviour and intensity of transmission, an approach that is most often not cost-effective. This study presents a detailed entomological description of mosquito distribution and variation in potentially transmissible contacts of Plasmodium falciparum following a south to north transect in Benin. METHOD: The study was conducted in five locations where environmental parameters were different and malaria prevalence ranged between 14 and 51%. The locations represent the main eco-epidemiological malaria areas in Benin. Mosquitoes were collected using human landing catches, pyrethrum spray catches and windows traps. They were taxonomically and molecularly identified. Head-thoraces of Anopheles gambiae s.l. were tested by enzyme-linked immunosorbent assay. Entomological indicators were estimated following WHO guidelines. RESULTS: The results showed variation between location and period in distribution of Anopheles coluzzii, An. gambiae, and Anopheles arabiensis (p < 0.05). An extension of the reported range of An. arabiensis was also observed. Densities of malaria vectors varied significantly between rural and urban sites, however, indoor/outdoor biting ratios remained constant. Proportions of malaria vectors with circumsporozoite protein of P. falciparum were similar between locations. The entomological inoculation rates ranged between zero and eight bites/man/night with significant variations between areas.Four profiles of human exposure to infectious malaria vector bites were observed and included location with one season of high transmission (June - August), two seasons of lower transmission (March-August; October-November), moderate continuous transmission season, and high continuous transmission season of P. falciparum. CONCLUSION: The study revealed several entomological patterns in transmission of P. falciparum in Benin. The data could be used for purposes of planning a more cost-effective vector control strategy, by stratifying the country into higher and lower transmission zones. The information could also be used to guide extension of indoor residual spray based on a targeted use of IRS at sites where the duration of insecticidal effect following spraying coincides with the peak transmission period.

Cryptic population structure and insecticide resistance in Anopheles gambiae from the southern Democratic Republic of Congo.

The Democratic Republic of Congo (DRC) suffers from one of the highest malaria burdens worldwide, but information on its Anopheles vector populations is relatively limited. Preventative malaria control in DRC is reliant on pyrethroid-treated nets, raising concerns over the potential impacts of insecticide resistance. We sampled Anopheles gambiae from three geographically distinct populations (Kimpese, Kapolowe and Mikalayi) in southern DRC, collecting from three sub-sites per population and characterising mosquito collections from each for resistance to pyrethroids using WHO tube bioassays. Resistance to each of three different pyrethroids was generally high in An. gambiae with < 92% mortality in all tests, but varied between collections, with mosquitoes from Kimpese being the most resistant. Whole genome sequencing of 165 An. gambiae revealed evidence for genetic differentiation between Kimpese and Kapolowe/Mikalayi, but not between the latter two sample sites despite separation of approximately 800 km. Surprisingly, there was evidence of population structure at a small spatial scale between collection subsites in Kimpese, despite separation of just tens of kilometres. Intra-population (H12) and inter-population (FST) genome scans identified multiple peaks corresponding to genes associated with insecticide resistance such as the voltage gated sodium channel (Vgsc) target site on chromosome 2L, a Cyp6 cytochrome P450 cluster on chromosome arm 2R, and the Cyp9k1 P450 gene on chromosome X. In addition, in the Kimpese subsites, the P450 redox partner gene Cpr showed evidence for contemporary selection (H12) and population differentiation (FST) meriting further exploration as a potential resistance associated marker.

Evidence of intensification of pyrethroid resistance in the major malaria vectors in Kinshasa, Democratic Republic of Congo.

Assessing patterns and evolution of insecticide resistance in malaria vectors is a prerequisite to design suitable control strategies. Here, we characterised resistance profile in Anopheles gambiae and Anopheles funestus in Kinshasa and assess the level of aggravation by comparing to previous 2015 estimates. Both species collected in July 2021 were highly resistant to pyrethroids at 1×, 5× and 10× concentrations (mortality < 90%) and remain fully susceptible to bendiocarb and pirimiphos methyl. Compared to 2015, Partial recovery of susceptibility was observed in A. gambiae after PBO synergist assays for both permethrin and α-cypermethrin and total recovery of susceptibility was observed for deltamethrin in 2021. In addition, the efficacy of most bednets decreased significantly in 2021. Genotyping of resistance markers revealed a near fixation of the L1014-Kdr mutation (98.3%) in A. gambiae in 2021. The frequency of the 119F-GSTe2 resistant significantly increased between 2015 and 2021 (19.6% vs 33.3%; P = 0.02) in A. funestus. Transcriptomic analysis also revealed a significant increased expression (P < 0.001) of key cytochrome P450s in A. funestus notably CYP6P9a. The escalation of pyrethroid resistance observed in Anopheles populations from Kinshasa coupled with increased frequency/expression level of resistance genes highlights an urgent need to implement tools to improve malaria vector control.

Data from Entomological Collections of Aedes (Diptera: Culicidae) in a post-epidemic area of Chikungunya, City of Kinshasa, Democratic Republic of Congo.

Arbovirus epidemics (chikungunya, dengue, West Nile fever, yellow fever and zika) are a growing threat in African areas where Aedes (Stegomyia) aegypti (Linnaeus, 1762) and Aedes albopictus (Skuse, 1895) are present. The lack of comprehensive sampling of these two vectors limits our understanding of their propagation dynamics in areas at risk of arboviruses. Here, we collected 6,943 observations (both larval and human capture) of Ae. aegypti and Ae. albopictus between 2020 and 2022. The study was carried out in the Vallee de la Funa, a post-epidemic zone in the city of Kinshasa, Democratic Republic of Congo. Our results provide important information for future basic and advanced studies on the ecology and phenology of these vectors, as well as on vector dynamics after a post-epidemic period. The data from this study are published in the public domain as the Darwin Core Archive in the Global Biodiversity Information Facility.

An evidence synthesis approach for combining different data sources illustrated using entomological efficacy of insecticides for indoor residual spraying.

BACKGROUND: Prospective malaria public health interventions are initially tested for entomological impact using standardised experimental hut trials. In some cases, data are collated as aggregated counts of potential outcomes from mosquito feeding attempts given the presence of an insecticidal intervention. Comprehensive data i.e. full breakdowns of probable outcomes of mosquito feeding attempts, are more rarely available. Bayesian evidence synthesis is a framework that explicitly combines data sources to enable the joint estimation of parameters and their uncertainties. The aggregated and comprehensive data can be combined using an evidence synthesis approach to enhance our inference about the potential impact of vector control products across different settings over time. METHODS: Aggregated and comprehensive data from a meta-analysis of the impact of Pirimiphos-methyl, an indoor residual spray (IRS) product active ingredient, used on wall surfaces to kill mosquitoes and reduce malaria transmission, were analysed using a series of statistical models to understand the benefits and limitations of each. RESULTS: Many more data are available in aggregated format (N = 23 datasets, 4 studies) relative to comprehensive format (N = 2 datasets, 1 study). The evidence synthesis model had the smallest uncertainty at predicting the probability of mosquitoes dying or surviving and blood-feeding. Generating odds ratios from the correlated Bernoulli random sample indicates that when mortality and blood-feeding are positively correlated, as exhibited in our data, the number of successfully fed mosquitoes will be under-estimated. Analysis of either dataset alone is problematic because aggregated data require an assumption of independence and there are few and variable data in the comprehensive format. CONCLUSIONS: We developed an approach to combine sources from trials to maximise the inference that can be made from such data and that is applicable to other systems. Bayesian evidence synthesis enables inference from multiple datasets simultaneously to give a more informative result and highlight conflicts between sources. Advantages and limitations of these models are discussed.

ImergardTMWP: A Non-Chemical Alternative for an Indoor Residual Spray, Effective against Pyrethroid-Resistant Anopheles gambiae (s.l.) in Africa.

Malaria is the deadliest mosquito-borne disease and kills predominantly people in sub-Saharan Africa (SSA). The now widespread mosquito resistance to pyrethroids, with rapidly growing resistance to other insecticide classes recommended by the World Health Organization (WHO), may overturn the successes gained in mosquito control in recent years. It is of utmost importance to search for new, inexpensive, and safe alternatives, with new modes of action, that might improve the efficacy of current insecticides. The efficacy of a novel mechanical insecticidal mineral derived from volcanic rock, ImergardTMWP, was investigated to determine its efficacy as a stand-alone residual wall spray and as a mixture with deltamethrin (K-Othrine® Polyzone) in experimental huts in Cove, Benin. The evaluation was conducted with susceptible (Kisumu) and wild-type Anopheles gambiae (s.l.). Deltamethrin applied alone demonstrated 40-45% mortality (at 72 h post-exposure) during the first four months, which declined to 25% at six months for wild An. gambiae from Cove. ImergardTMWP alone and mixed with deltamethrin, under the same assay conditions, produced 79-82% and 73-81% mortality, respectively, during the same six-month period. ImergardTMWP met the 80% WHO bio-efficacy threshold for residual activity for the first five months with 78% residual activity at six months. ImergardTMWP can be used as a mixture with chemical insecticides or as a stand-alone pesticide for mosquito control in Africa.

Genetic structure of Anopheles gambiae s.s populations following the use of insecticides on several consecutive years in southern Benin.

BACKGROUND: Several studies have reported the strong resistance of Anopheles gambiae s.l. complex species to pyrethroids. The voltage-dependent sodium channel (Vgsc) gene is the main target of pyrethroids and DDT. In Benin, the frequency of the resistant allele (L1014F) of this gene varies along the north-south transect. Monitoring the evolution of resistance is necessary to better appreciate the genetic structure of vector populations in localities subject to the intensive use of chemicals associated with other control initiatives. The purpose of this study was to map the distribution of pyrethroid insecticide resistance alleles of the Kdr gene in malaria vectors in different regions and ecological facies in order to identify the evolutionary forces that might be the basis of anopheline population dynamics. METHODS: The characterization of Anopheles gambiae s.l. populations and resistance mechanisms were performed using adult mosquitoes obtained from larvae collected in the four agroecological zones in southern Benin. Genomic DNA extraction was performed on whole mosquitoes.The extracted genomic DNA from them were used for the molecular identification of species in Anopheles gambiae s.l. complex and the identification of genotypes related to pyrethroid resistance as the Kdr gene amino acid position 1014 in sodium channel. Molecular speciation and genotyping of Kdr resistant alleles (1014) were done using PCR.Genepop software version 4.2 was used to calculate allelic and genotypic frequencies in each agroecological zone. The p value of the allelic frequency was determined using the binomial test function in R version 3.3.3. The Hardy-Weinberg equilibrium was checked for each population with Genetics software version 1.3.8.1. The observed heterozygosity and the expected heterozygosity as well as the fixation index and genetic differentiation index within and between populations were calculated using Genepop software version 4.2. RESULTS: During the study period, Anopheles coluzzii was the major species in all agroecological zones while Anopheles gambiae was scarcely represented. Regardless of the species, resistant homozygote individuals (L1014F/L1014F) were dominant in all agroecological zones, showing a strong selection of the resistant allele (L1014F). All populations showed a deficit of heterozygosity. No genetic differentiation was observed between the different populations of the two species. For Anopheles coluzzii, there was a small differentiation among the populations of the central cotton and bar-lands zones. The genetic differentiation was modest among the population of the fisheries zone (Fst = 0.1295). The genetic differentiation was very high in the population of Anopheles gambiae of the bar-lands zone (Fst = 0.2408). CONCLUSION: This study revealed that the use of insecticides in Benin for years has altered the genetic structure of Anopheles gambiae s.s. populations in all agroecological zones of southern Benin. It would be desirable to orientate vector control efforts towards the use of insecticides other than pyrethroids and DDT or combinations of insecticides with different modes of action.

Efficacy of Actellic 300 CS-based indoor residual spraying on key entomological indicators of malaria transmission in Alibori and Donga, two regions of northern Benin.

BACKGROUND: The current study shows the results of three years of IRS entomological monitoring (2016, before intervention; 2017 and 2018, after intervention) performed in Alibori and Donga, northern Benin. METHODS: Mosquito collections were performed on a monthly basis using human landing catches and pyrethrum spray catches in six districts including four treated with Actellic 300 CS (Kandi, Gogounou, Djougou and Copargo) and two untreated (Bembèrèkè and Kouandé) which served as control sites. Key transmission indicators of Anopheles gambiae (s.l.) as well as the residual activity of Actellic 300 CS assessed through WHO cone tests, were determined. RESULTS: The residual efficacy duration of Actellic 300 CS after the two IRS campaigns (2017 and 2018) was 4-5 months (May-September). The parity rate and the sporozoite index of An. gambiae (s.l.) were 36.62% and 0.71%, respectively, after the first spray round in treated areas compared to 57.24% and 3.7%, respectively, in the control areas (P < 0.0001). The same trend was observed after the second spray round. After the first spray round, each person received 1.6 infective bites/month (ib/m) in the treated areas against 12.11 ib/m in the control areas, resulting in a reduction rate of 86.78%. Similarly, the entomological inoculation rate was 1.5 ib/m after the second spray round in the treated areas vs 9.75 ib/m in the control areas, corresponding to a reduction of 84.61%. A decrease in the parity rate (46.26%), sporozoite index (85.75%) and EIR (87.27%) was observed for An. gambiae (s.l.) after the first round of IRS (June-October 2017) compared to the pre-intervention period (June-October 2016). The density of An. gambiae (s.l.) ranged between 0.38-0.48 per house in treated areas vs 1.53-1.76 An. gambiae (s.l.) per house respectively after the first and second IRS rounds. CONCLUSIONS: This study showed the positive impact of IRS in reducing key entomological parameters of malaria transmission in Alibori and Donga. However, the considerable blood-feeding rate of An. gambiae (s.l.) in spray areas, stress the need for the population to sleep under long-lasting insecticidal nets (LLINs) in addition, to prevent from mosquito bites which did not succeed in resting on sprayed walls.