Profile and Characterization of Insecticide Resistance Mechanisms in the Anopheles gambiae s.l. Population of Sierra Leone

Profile and Characterization of Insecticide Resistance Mechanisms in the Anopheles gambiae s.l. 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Population of Sierra Leone Kevin Ochieng Opondo, Evelyne Alyko, Samuel Smith, Rebecca Levine, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4720552/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Mar, 2025 Read the published version in Malaria Journal → Version 1 posted 10 You are reading this latest preprint version Abstract Background Information on the status of insecticide resistance in malaria vectors is critical for implementing effective malaria vector control. The Sierra Leone National Malaria Control Program, in collaboration with the PMI VectorLink project, assessed the resistance status to insecticides commonly used in public health, and associated resistance mechanisms in Anopheles gambiae s.l., the main vector of malaria in Sierra Leone. Methods The susceptibility of An. gambiae s.l. against pyrethroids with and without piperonyl butoxide (PBO), chlorfenapyr, clothianidin, bendiocarb and pirimiphos-methyl was evaluated in four districts of Sierra Leone in 2018 and 2019 using WHO and CDC bottle bioassay protocols. A subset of samples that were exposed to the insecticides were screened for molecular markers of insecticide resistance, knock-down resistance (kdr) L1014F, 1014S and N1575Y, and ( Ace-1 -G119S). Results Anopheles gambiae s.l. from all sites were resistant to the diagnostic doses of three pyrethroids: deltamethrin, permethrin and alpha-cypermethrin. Intensity of resistance to all three pyrethroids was high, with less than 95% mortality at 10X concentration. However, pre-exposure of An. gambiae s.l. to PBO increased overall mortality by 41.6%, 50.0% and 44.0% for deltamethrin, permethrin and alpha-cypermethrin, respectively. The vector was susceptible to chlorfenapyr, clothianidin and pirimiphos-methyl, while bendiocarb showed possible resistance. The frequency of kdr alleles was 98.2% for L1014F, 2.1% for 1014S and 8.9% for N1575Y, while the frequency of the Ace-1 G119S allele was 13.6%. Significant deviation from the Hardy-Weinberg equilibrium and deficiency of heterozygotes was detected only at the G119S locus of An. gambiae s.l. (p < 0.0001). Of the 191 An. gambiae s.l. that were molecularly identified to the species level, 81.7% were An. gambiae (95% CI 75.3–86.7), followed by An. coluzzii (17.8%, 95% CI (12.8–24.1) with one hybrid of An. gambiae/An. coluzzii 0.5%, 95% CI (0.03–3.3). Conclusion Malaria vectors were highly resistant to pyrethroids but exposure to PBO partially restored susceptibility in An. gambiae s.l. in Sierra Leone. Malaria vectors were susceptible to chlorfenapyr, clothianidin and pirimiphos-methyl with possible resistance to bendiocarb. These data informed the selection and distribution of ITN PBO in Sierra Leone’s mass campaigns in 2020 and selection of clothianidin for indoor residual spraying in 2021. Insecticide resistance Anopheles gambiae s.l. kdr Ace-1 Sierra Leone Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Globally, an estimated 247 million cases of malaria and 619,000 deaths occurred in 2021 with 95% of the cases reported from Africa [ 1 ]. The majority (96%) of malaria deaths occurred in Africa with children under five years of age accounting for 80% of the malaria deaths [ 1 , 2 ]. Between 2000 and 2015, global malaria case incidence declined by 27%, and by less than 2% between 2015 and 2019, and stagnated since 2021 indicating a slowing of the rate of decline since 2015 [ 1 , 3 ]. Indoor residual spraying (IRS) and insecticide treated nets (ITNs) are considered core interventions in the global control of malaria, which have contributed to the decline in malaria cases globally over the last two decades [ 1 , 3 ]. However, the intensive use of insecticides in agriculture and to a lesser degree in public health has led to widespread resistance among targeted mosquito vectors [ 4 – 6 ] and the major gains in the control of malaria have been threatened due to insecticide resistance [ 4 ]. Malaria is endemic in Sierra Leone, with stable and perennial transmission throughout the country. It is the leading cause of morbidity and mortality and accounts for 40.3% of all-age outpatient morbidity, 37.6% of hospitalizations, and has a case fatality rate of 17.6% in children under five years of age [ 7 ]. The Sierra Leone National Malaria Control Program (NMCP) conducted mass distribution of ITNs in 2010, 2014, 2017 and 2020 to achieve universal coverage. The mass distribution in May 2020 was conducted solely with ITNs impregnated with a pyrethroid and the synergist piperonyl-butoxide (PBO ITNs), making Sierra Leone the world’s first country to deploy PBO ITNs nationwide. The next mass distribution campaign using PBO ITNs and Interceptor G2 (IG2, active ingredients of chlorfenapyr and alpha-cypermethrin) ITNs is planned to begin in January 2024. The mass campaigns are complemented by routine IG2 or PBO ITN distribution through antenatal care (ANC) and Expanded Program on Immunization (EPI) clinics including during Maternal and Child Health Week. A pilot IRS campaign with lambda-cyhalothrin was implemented in four districts in 2011 and 2012, covering 5.8% of the population in Sierra Leone. In 2021, the NMCP in collaboration with the U.S. President’s Malaria Initiative (PMI) began implementing IRS with SumiShield™ (active ingredient, clothianidin) in Bo and Bombali districts that had the highest entomological inoculation rates (EIR) of over 410 infectious bites/person/year with malaria prevalence of between 38 and 40% in children under 5 years [ 7 – 9 ]. Successful implementation of a vector control program relies on knowledge of vector species, behavior, infectivity, and susceptibility to insecticides [ 10 ]. Members of the Anopheles gambiae complex and An. funestus group are the major vectors of malaria in sub-Saharan Africa (SSA), including Sierra Leone [ 11 , 12 ]. Prior to this study, An. gambiae s.s. and An. coluzzii had been reported in Sierra Leone but their insecticide resistance status was not well established due to the disruption of entomological studies during the civil war (from 1991 to 2002) and beyond [ 13 ]. Characterization of insecticide resistance in mosquitoes was later reported, but only in urban Freetown [ 14 ]. The knock-down resistance (kdr) mutation conferring resistance to pyrethroids and Dichlorodiphenyltrichloroethane (DDT), first reported in 1991 in An. gambiae s.s. populations in Côte d’Ivoire [ 15 ], has spread within the species complex within SSA, including to Sierra Leonen[ 4 , 14 ]. Although some studies have indicated that ITNs remain effective for providing protection in areas with pyrethroid resistant vectors [ 16 – 19 ], other studies suggest that in areas where there is high levels of kdr in local vector populations, pyrethroid treated ITNs or pyrethroid-based IRS products failed to control the An. gambiae s.l. population [ 20 , 21 ]. The new generation ITNs incorporating PBO or dual active ingredients have been shown to be more effective against pyrethroid resistant mosquitoes [ 22 ]. To better guide deployment of vector control interventions in Sierra Leone, the NMCP in collaboration with PMI VectorLink project assessed the susceptibility of malaria vectors to common insecticides used in malaria control as well as the distribution of molecular markers of insecticide resistance between 2018–19. We investigated the status of phenotypic resistance to insecticides in the pyrethroid, organophosphate, carbamate, neonicotinoid, and pyrrole classes of insecticides and determined the occurrence and frequency of molecular mechanisms for pyrethroid, organophosphate and carbamate resistance. The aims were to characterize resistance of the An. gambiae complex mosquitoes to insecticides currently in use and under consideration for use in IRS and ITNs, to inform decision making for optimizing vector control. Methods Sampling area Sierra Leone is located on the west coast of Africa, bordered on the north and east by Guinea, on the south by Liberia, and opens into the Atlantic Ocean to the West. The study was conducted in four sites in four districts (Bo, Bombali, Kono and Western Rural Area) representing the different geographical regions of Sierra Leone. Bo represents the southern region; Bombali the northern; Kono the eastern; and Western Area Rural the west. All the districts lie within the rainforest belt vegetation with Western Area Rural having a coastal ecosystem with brackish water due to the presence of Atlantic Ocean. The rainfall pattern is similar in all sites, beginning in May and ending in October. The dry season begins in November through to April. In each district, mosquitoes were collected from one rural chiefdom: Jaima Bongor in Bo, Gbanti Kamaranka in Bombali, Nimyama in Kono and Koya in Western Area Rural (Fig. 1 ). The four sites are also where the PMI VectorLink project in Sierra Leone has been doing comprehensive entomological monitoring activities since 2018. Insecticide susceptibility tests World Health Organization (WHO) tube tests [ 23 ] and Centers for Disease Control and Prevention (CDC) bottle bioassays [ 24 ] were performed to assess the susceptibility of An. gambiae s.l. to the most common insecticides used in ITNs and IRS in Sierra Leone. The WHO impregnated test papers at diagnostic doses (1x) of pyrethroids (alpha-cypermethrin (0.05%), deltamethrin (0.05%) and permethrin (0.75%), pirimiphos methyl (0.25%) and bendiocarb (0.1%) were used to assess mosquito susceptibility. The WHO tube method was used to test An. gambiae s.l., susceptibility to pirimiphos methyl (0.25%) in all four sites and to bendiocarb (0.1%) in all sites except in Bombali, where insufficient mosquitoes were collected. The CDC bottle assay was used to assess the intensity of resistance to pyrethroids. Larvae and pupae of Anopheles mosquitoes were collected from breeding sites, mainly from temporary rain pools, in and around the sentinel sites and reared to adults at the Vector-Borne Disease Insectary and Laboratory (VBDIL) in Makeni, Bombali District. Mosquitoes were morphologically identified at the adult stage and only An. gambiae s.l. aged between 2–5 days old were used for the susceptibility tests. Tests were conducted in all four districts: Bo, Bombali, Kono and Western Rural Area. The number of dead and alive mosquitoes in both the exposure and the control tubes were recorded after a 24-hour post-exposure holding period. Given the slow-acting nature of clothianidin, post-exposure mortalities were scored every 24 hours for up to five days or until 100% mortality was recorded, whichever came first. The CDC bottle bioassay was also used to assess susceptibility of An. gambiae s.l. to chlorfenapyr. Two-to-five-day old An. gambiae s.l. reared from larvae were exposed to 250ml Wheaton bottles treated with a diagnostic concentration of 100µg/bottle chlorfenapyr. For clothianidin, the bottles were coated with 2% clothianidin. Tests with An. gambiae Kisumu as positive controls and negative controls without insecticide were run in parallel. Female Anophele s mosquitoes were introduced in batches of 20–25 in each replicate. After a 60 minutes exposure period, mosquitoes were released into clean cages and then gently aspirated into labeled paper cups covered with untreated netting and provided with 10% sugar solution. Knock-down was recorded 60 minutes after the start of the test, while mosquitoes were still in the bottle. Mortality was recorded every 24 hours for up to five days or until 100% mortality was recorded, whichever came first. Synergist, and intensity assays of pyrethroids First, mosquitoes were exposed to treated WHO papers at diagnostic doses (1x) of pyrethroids (alpha-cypermethrin (0.05%), deltamethrin (0.05%) and permethrin (0.75%)) with and without piperonyl butoxide 4% (PBO) synergist using the WHO procedure [ 23 ]. Adult An. gambiae s.l. mosquitoes aged 2–5 days old were pre-exposed to PBO for an hour followed by exposure to either deltamethrin, permethrin and alpha-cypermethrin impregnated papers (1x) in WHO tube tests. The number of dead and alive mosquitoes in both the exposure and the control tubes were recorded after a 24-hour post-exposure holding period. The mortality estimates in the insecticide-only group, without pre-exposure to PBO, at diagnostic doses were also taken as 1x in the intensity assays. The CDC bottle bioassay [ 24 ] was used to assess the intensity of resistance to pyrethroids above the diagnostic dose. Anopheles gambiae s.l. adult mosquitoes were exposed to alpha-cypermethrin and deltamethrin at 62.5 (5x) and 125 (10x) µg/bottle. Permethrin was tested at a dose of 107.5 (5x) and 215 (10x) µg/bottle. These intensities were estimated in all sites except Kono, where the number of larvae collected was only enough to measure the intensity of alpha-cypermethrin resistance. Molecular species identification A subset of 200 mosquitoes out of 5,326, composed of those that survived or died following insecticide exposures, and representing all the districts that were tested were randomly sampled for molecular analysis. Genomic DNA was extracted from each mosquito sample via the Livak method [ 25 ] and stored at -20ºC following extraction. Identification of members of the An. gambiae complex was done using SINE polymerase chain reaction (PCR) that allows identification of An. gambiae s.s., An. coluzzii and An. arabiensis [ 26 ]. In coastal sites where other species such as An. melas were suspected, the PCR-restriction fragment length polymorphism (PCR-RFLP) protocol described by Scott et al. [ 27 ] was used. PCR products were run via electrophoresis through a 1.5% agarose gel with Midori Green® (Gene flow, UK) and visualized using ultraviolet light. Molecular characterization of resistance mechanisms The subset samples above were also screened for the presence of the 1014F, 1014S, N1575Y, Ace-1G119S mutations using TaqMan assays [ 28 – 30 ]. Genotypes were scored from scatter plots of results produced by the Mx3005 v4.10 software. Three positive samples of known genotypes for each of the alleles were used as positive controls while distilled water was used as negative control for each of the experiments as described in the protocols ([ 28 – 31 ]. Data analysis Mortality was calculated by dividing the number of dead mosquitoes following exposure by total number exposed for each insecticide. Mortalities were corrected using Abbott’s formula if the mortality of control mosquitoes exposed to solvent only was ≥ 5% and 10%. Susceptibility levels of An. gambiae s.l. were evaluated based on the WHO criteria of test mortality [ 23 ]: corrected mortality of 98–100% after 24–120 hours post exposure indicated susceptibility; corrected mortality, > 90% but < 98%, indicated the existence of possible resistance; and mortality of < 90% indicated the presence of resistant individuals in the vector population. Genotype frequencies per species per site was calculated as the relative frequency of the homozygote resistant and heterozygote resistant individuals. The allelic frequencies of L1014F, L1014S, N1575Y and Ace-1 were calculated as follows: F(R) = [2RR + RS]/ [2(RR + RS + SS)]. The Hardy-Weinberg equation was used to calculate the expected genotype frequency of L1014F, L1014S, N1575Y and Ace-1 in An. gambiae s.s. and An. coluzzii. The expected and observed genotype frequencies were compared using Pearson’s Chi-squared tests in Microsoft Excel 2016 to determine statistical significance of differences and estimate inbreeding co-efficient (F IS ) and STATA-SE12 was used to generate confidence intervals for the resistance allelic frequency distributions. Results Insecticide susceptibility and intensity assays A total of 5,326 An. gambiae s.l. were successfully reared to adults and exposed to different insecticides (supplementary material Table 1). Anopheles gambiae s.l. from all sampling sites were resistant to the diagnostic dose of deltamethrin, permethrin and alpha-cypermethrin (Fig. 2 , supplementary material Table 1). The 24-hour post-exposure mortality ranged from 7% in Bombali to 65% in Western Area Rural for permethrin; from 12% in Bombali to 53% in Kono for deltamethrin; and from 10% in Bo to 50% in Western Area Rural for alpha-cypermethrin (Fig. 2 ). Anopheles gambiae s.l. was fully susceptible to pirimiphos-methyl in all sites. For bendiocarb, there was resistance in Western Area Rural with 88% mortality and possible resistance in Bo and Kono with 94% mortality. Resistance intensity to alpha-cypermethrin, deltamethrin and permethrin was high at all sites, with mortality rates of An. gambiae s.l. below 98% at 10 times (10x) the diagnostic dose (Fig. 2 ). Mortality was below 90% at five times (5x) the diagnostic dose for all the three pyrethroids except for alpha-cypermethrin in Bombali (Fig. 2 ). Anopheles gambiae s.l. was susceptible to chlorfenapyr in all four sampling sites (Fig. 3 ). In all sites, over 98% of mosquitoes exposed to chlorfenapyr died after 72-hours post-exposure with mortality reaching 100% after 120 hours (4 days post exposure) (Fig. 3 ). Anopheles gambiae s.l. was also fully susceptible to clothianidin at all sites (Fig. 3 ). In Western Rural Area and Bombali, 100% mortality to clothianidin was recorded within the 24-hour holding period. However, it took 72 hours to achieve 100% mortality in Bo and 120 hours in Kono (Fig. 3 ). Overall, mosquitoes died faster after exposure to clothianidin than to chlorfenapyr. At 24 hours holding time, the mean percent mortality was significantly higher (ꭓ 2 = 272.5; p < 0.0001) for clothianidin (94.1%) than for chlorfenapyr (35.9%) (Table 1 ). The difference narrowed at 48 hours holding time, but it was still significantly higher (ꭓ 2 = 4.8; p = 0.028) for clothianidin than for chlorfenapyr. There was no difference at 72 hours holding time (ꭓ 2 = 1.2; p = 0.273) and beyond (Table 1 ). Table 1 Mean percent mortality of An. gambiae s.l. by holding time post exposure to chlorfenapyr and clothianidin. Holding time Mean mortality 24hrs 48hrs 72hrs 96hrs 120hrs Chlorfenapyr 35.9 95.8 99.2 99.5 100 Clothianidin 94.1 98.5 99.7 99.7 100 Test ꭓ 2 = 272.5 ꭓ 2 = 4.8 ꭓ 2 = 0.8 ꭓ 2 = 0.2 NA P value p = < 0.0001 p = 0.028 p = 0.273 p = 0.644 NA Synergist assays Exposure to 4% PBO increased mortality for each pyrethroid insecticide but full restoration of susceptibility above 98% was not achieved for any insecticides in any locations. The absolute increase after pre-exposure for deltamethrin ranged from 43.9% in Bombali to 52.9% in Bo. For alpha-cypermethrin, the absolute increase ranged between 41.7% in Bo and 56.0% in Kono. The absolute increase for permethrin was highest in Kono (66.3%) and lowest in Western Rural Area (41.5%) (Fig. 4 ). Molecular species identification A total of 191 out of 200 (95.5%) mosquitoes were successfully identified to sibling species. Anopheles gambiae was dominant (81.7% 95% CI (75.3–86.7), followed by An. coluzzii (17.8%, 95% CI (12.8–24.1). One specimen was identified as a hybrid of An. gambiae and An. coluzzii 0.5%, 95% CI (0.03–3.3). Molecular characterization of resistance mechanisms The frequency of the kdr L1014F resistance (r) allele was high, 0.98 (373/380); 95% CI: 0.94–0.99) whereas the kdr L1014S mutation frequency was low at 0.02 (8/382); 95% CI: 0.01–0.04) across all four assessment sites (Table 3 ). The wild-type allele for kdr L1014F was not detected in any of the An. gambiae or An. coluzzii specimens screened (Tables 2 and 3 ). The N1575Y mutation, which amplifies resistance conferred by L1014F and L1014S, was detected at a frequency of 0.089 (34/384) and were all in a heterozygous form (Tables 2 and 3 ). The difference in the frequency of the N1575Y mutation between An. gambiae (0.095) and An. coluzzii (0.059) was not statistically significant (p = 0.928). All 34 samples with the N1575Y mutation also carried the homozygous mutant allele L1014F. No deviation from the Hardy-Weinberg equilibrium and no deficiency of heterozygous individuals was detected at the L1014F, L1014S and N1575Y loci (Tables 3 and 4 ). Table 2: Distribution of molecular markers of insecticide resistance by phenotype in Sierra Leone Kdr-w Kdr-e Ace-1 N1575Y District Phenotype FF LF NA Total LS LL NA Total SS GG NA Total NY NN NA Total Bo Resistant 31 2 33 4 29 33 4 29 33 3 30 33 Susceptible 17 17 1 15 1 17 1 16 17 2 15 17 Bombali Resistant 31 31 3 28 31 31 31 8 23 31 Susceptible 19 1 20 19 1 20 3 16 1 20 5 14 19 Kono Resistant 27 2 2 31 30 1 31 3 27 1 31 4 27 31 Susceptible 18 1 19 1 18 19 2 17 19 1 18 19 Western Rural Area Resistant 31 31 31 31 12 19 31 8 23 31 Susceptible 17 2 19 18 1 19 1 18 19 4 14 2 20 Total 191 7 3 201 9 188 4 201 26 173 2 201 35 164 201 NA = Not amplified; LL = homozygous wild type; LF = heterozygous resistant for kdr-w ; FF = homozygous resistant for kdr-w ; LS= heterozygous resistant for kdr-e ; GG= homozygous wild type; SS = homozygous resistant for Ace -1; NN = homozygous wild type for N1575Y; NY = heterozygous resistant for N15756Y. Table 3 Allelic frequencies of L1014F and L1014S Site Species L1014 N LL LF FF f (L1014F) 95% CI F IS N LL LS SS f(L1014S) 95% CI F IS Bo An. coluzzii 17 0 1 16 0.97 (0.85-1.00) -0.03030 17 16 1 0 0.03 (0.00-0.15) -0.0303 An. gambiae 30 0 1 29 0.98 (0.91-1.00) -0.01695 30 27 3 0 0.05 (0.01–0.14) -0.05263 Bombali An. coluzzii 6 0 0 6 1.00 (0.74-1.00) - 6 6 0 0 0.00 (0.00-0.27) - An. gambiae 44 0 0 44 1.00 (96.0–1.00) - 44 41 3 0 0.03 (0.01–0.10) -0.03529 Kono (Eastern) An. coluzzii 4 0 1 3 0.88 (0.47-1.00) -0.14286 4 3 1 0 0.01 (0.00-0.53) -0.14286 An. gambiae 42 0 2 40 0.98 (0.92-1.00) -0.02439 43 43 0 0 0.00 (0.00-0.04) - Western Rural Area An. coluzzii 7 0 2 5 0.86 (0.57–0.98) -0.16667 7 7 0 0 0.00 (0.00-0.23) - An. gambiae 40 0 0 40 1.00 (95.0–1.00) - 40 40 0 0 0.00 (0.00-0.05) - Total 190 0 7 183 0.98 (0.94–0.99) -0.01799 191 183 8 0 0.02 (0.01–0.04) -0.02139 N = # tested; LL = homozygous wild type; LF = heterozygous resistant for kdr-w ; FF = homozygous resistant for kdr-w ; f (L1014F) = frequency of the L1014F resistant allele; LS = heterozygous resistant for kdr-e ; SS = homozygous resistant for kdr-e ; f (L1014S) = frequency of the L1014S resistant allele. F IS Values > 0 indicate heterozygote deficiency, while values < 0 indicate heterozygote excess. Table 4 Allelic frequencies of N1575Y and G119S Site N1575Y G119S Species N NN NY YY f(N1575Y) 95% CI F IS N GG GS SS f(G119S) 95% CI F IS Bo An. coluzzii 17 16 1 0 0.03 (0.00-0.15) -0.03030 17 17 0 0 0.00 (0.00-0.10) - An. gambiae 30 27 3 0 0.05 (0.01–0.14) -0.05263 30 25 0 5 0.17 (0.08–0.29) 1.000* Bombali An. coluzzii 6 4 2 0 0.17 (0.02–0.48) -0.20000 6 6 0 0 0.00 (0.00-0.26) - An. gambiae 44 33 11 0 0.13 (0.06–0.21) -0.14286 44 41 0 3 0.07 (0.03–0.14) 1.000* Kono An. coluzzii 4 4 0 0 0.00 (0.00-0.37) - 4 4 0 0 0.00 (0.00-0.37) - An. gambiae 44 39 5 0 0.06 (0.02–0.13) -0.06024 43 38 0 5 0.12 (0.06–0.20) 1.000* Western Rural Area An. coluzzii 7 6 1 0 0.07 (0.00-0.34) -0.07692 7 7 0 0 0.00 (0.00-0.23) - An. gambiae 40 29 11 0 0.14 (0.07–0.23) -0.15942 40 27 0 13 0.33 (0.22–0.44) 1.000* Total 192 158 34 0 0.09 (0.06–0.12) -0.09714 191 165 0 26 0.14 (0.01–0.17) 1.000 N = # tested; GG = homozygous wild type; GS = heterozygous resistant for Ace -1; SS = homozygous resistant for Ace -1; f (G119S) = frequency of the G119S resistant allele; NN = homozypous wild type for N1575Y; NY = heterozygous resistant for N15756Y; YY = homozygous resistant for N1575Y; f (N1575Y) = frequency of the N1575Y resistant allele. F IS values > 0 indicate heterozygote deficiency, while values < 0 indicate heterozygote excess. The overall frequency of the G119S mutation was 0.14 (N = 52). No G119S mutation was found in An. coluzzii (Table 4 ). However, both deviation from the Hardy-Weinberg equilibrium and deficiency of heterozygotes was detected at the G119S locus of An. gambiae s.s. and was statistically significant (p < 0.0001) (Table 5 ). As all the samples were either homozygous or heterozygous to L1014F mutation, all the specimens with L1014S, G119S and N1575Y also had the L1014F resistance allele. All mosquitoes with L1014S did not have the G119S mutation and only one was carrying the N1575Y mutation. Seven An. gambiae s.l. specimens were found to be carrying three mutations: L1014F plus either L1014S or G119S, or L1014F plus G119S and N1575Y (Table 6 ). Table 5 Genotype frequencies for the L1014 kdr, G119S, and N1575Y mutation in An. gambiae s.l. from Sierra Leone. Tests of Hardy-Weinberg Equilibrium (χ2) with corresponding P-values. Mutation Site N Observed χ2 P-VALUE RR RS SS L1014F Bo (Southern) 50 0.960 0.040 0.000 0.0208 0.8853 Bombali (Northern) 50 1.000 0.000 0.000 0.0000 1.0000 Western Rural Area (Western) 50 0.960 0.040 0.000 0.0208 0.8853 Kono (Eastern) 48 0.938 0.063 0.000 0.0499 0.8232 L1014S Bo (Southern) 49 0.000 0.102 0.898 0.1416 0.7067 Bombali (Northern) 50 0.000 0.060 0.940 0.0478 0.8269 Western Rural Area (Western) 49 0.000 0.000 1.000 0.0000 1.0000 Kono (Eastern) 49 0.000 0.020 0.980 0.0052 0.9425 G119S Bo (Southern) 50 0.100 0.000 0.900 50.0000 < .00001 Bombali (Northern) 50 0.060 0.000 0.940 50.0000 < .00001 Western Rural Area (Western) 50 0.260 0.000 0.740 50.0000 < .00001 Kono (Eastern) 49 0.102 0.000 0.898 49.0000 < .00001 N1575Y Bo (Southern) 50 0.000 0.100 0.900 0.1385 0.7098 Bombali (Northern) 50 0.000 0.260 0.740 1.1164 0.2907 Western Rural Area (Western) 49 0.000 0.245 0.755 0.9540 0.3287 Kono (Eastern) 50 0.000 0.100 0.900 0.1385 0.7098 Table 6 Multiple mutations in An. gambiae s.l. Mutations L1014F L1014S G119S N1575Y Phenotype Resistant Susceptible Resistant Susceptible Resistant Susceptible Resistant Susceptible L1014F -- -- -- -- -- 23 11 L1014S 6 2 -- -- -- -- 1 0 G119S 19 7 0 -- -- -- 6 0 N1575Y 23 11 1* -- 6* -- -- -- *Also carry the L1014F mutation; 7 samples were with 3 types of mutations Seven of the eight samples with the G119S mutation were also survivors of the exposure to bendiocarb. However, 68.2% (15/22) of the samples that were phenotypically resistant to bendiocarb were not carrying the G119S mutation. Ninety-five percent (19/20) of the samples susceptible to bendiocarb and pirimiphos-methyl were also not carrying the mutation (Table 7 ). Table 7 Phenotypic resistance of An. gambiae s.l. to bendiocarb and allelic frequencies of G119S Species Phenotype N G119S GG GS SS Odds ratio An. gambiae s.l. Resistant/Alive 22 15 0 7 7.7, p = 0.03 Susceptible/Dead 20 19 0 1 Ref Total 42 34 0 8 GG = homozygous wild type; GS = heterozygous resistant for Ace -1; SS = homozygous resistant for Ace -1 Discussion Anopheles gambiae s.s. was the dominant malaria vector in Sierra Leone followed by An. coluzzii . Consistent with previous studies, they were the only species sampled in larval habitats [ 13 ]. The main malaria vector in Sierra Leone, An. gambiae s.l., was highly resistant to the three pyrethroid insecticides tested with partial restoration of susceptibility following pre-exposure to PBO. The presence of survivors following exposure to 10x the discriminating doses is indicative of the high intensity of resistance to pyrethroids. The observed range of enhancement of mortality due to pre-exposure to PBO was similar across the three pyrethroids tested. This assessment also demonstrated the kdr L1014F resistance mutation to be present at high frequency, which is not surprising, considering the high level of phenotypic resistance to pyrethroids. Though not frequently identified in previous samples, this report is a first of kdr L1014S and N1575Y mutations in Sierra Leone. The N1575Y mutation, located in the Vgsc and believed to have a synergistic effect on pyrethroid and DDT resistance when combined with the L1014F mutation was previously reported from Burkina Faso, Benin, Cameroon and Côte d’Ivoire [ 31 , 33 – 35 ]. The detection of the N1575Y mutation in Sierra Leone requires more investigation to better characterize its expected synergistic relationship with 1014F kdr . The presence of this additional mechanism that could further reduce insecticide efficacy in the already pyrethroid-resistant mosquitoes in Sierra Leone is concerning. Thus, monitoring of N1575Y should continue in order to understand its contribution to insecticide resistance among local vectors. This resistance mechanism could spread very rapidly [ 31 ] and threaten the malaria vector control efforts in Sierra Leone which had relied primarily on the distribution of pyrethroid-treated ITNs. This finding also provides evidence for justifying the need to move to the distribution of non-pyrethroid nets. As the effect of PBO exposure was similar across the three pyrethroids tested, nets treated with any of the pyrethroids plus PBO might have similar effects and can be used as alternative tools in the national Sierra Leone Insecticide Resistance Management Plan. Indeed, NMCP distributed PermaNet 3.0 (deltamethrin + PBO) and Olyset Plus (permethrin + PBO) in the 2020 mass net campaign and these contributed to malaria reduction in Sierra Leone [ 36 ]. These data not only yielded the evidence that PBO nets could provide better protection than pyrethroid nets without PBO in Sierra Leone, but also demonstrated that in the absence of non-pyrethroid ITNs available at the time of this assessment, Sierra Leone had the flexibility to choose PBO ITNs treated with any of the three pyrethroids. Sierra Leone was the world’s first country to provide population-level coverage nationally with next-generation PBO ITNs during a mass distribution campaign and evaluations to measure their durability and performance are underway. The NMCP has now incorporated these data and subsequent data on insecticide resistance to procure IG2 nets for 2023 mass ITN campaign. Anopheles gambiae s.l. was susceptible to pirimiphos-methyl (organophosphate), chlorfenapyr (pyrrole) and clothianidin (neonicotinoid). However, there was an indication of possible resistance to bendiocarb (carbamate). As the NMCP prepared to implement IRS in Bo and Bombali districts in May/June 2021 at the start of the rainy season, the findings from this assessment provided critical data that guided the selection of an appropriate insecticide for the IRS program. Thus, clothianidin was selected for IRS because vectors were fully susceptible to it. There was no phenotypic resistance to pirimiphos-methyl and low-level resistance to bendiocarb was reported. However, carbamates and organophosphates are known to share the Ace - 1 pathway as a resistance mechanism [ 37 ]. With the reported phenotypic resistance to bendiocarb and the reported prevalence of the G119S mutation at 13.6% in the vector population, this mutation needs close monitoring to guide future IRS in Sierra Leone. Moreover, the combination of IRS using pirimiphos-methyl and PBO nets have been suggested to be antagonistic [ 38 ]. Thus, with the mass distribution of PBO nets that occurred in Sierra Leone in 2020 and continued PBO ITN distribution through some routine channels, and considering this potential antagonism between vector control interventions, caution should be used in the selection of pirimiphos-methyl for IRS. The susceptibility of the vector to chlorfenapyr also suggests that its use for IRS or as part of ITN treatment is a possibility when it becomes available. The presence of three or more Ace-1 alleles in An. gambiae s.s. mosquitoes was previously documented in several countries in West Africa [ 39 – 41 ]. However, prior to the present assessment, little information was available for Sierra Leone. Although the G119S mutation is indicative of the phenotypic resistance to bendiocarb in this study, the high number of wild types that survived exposure to bendiocarb indicates involvement of other resistance mechanisms in Sierra Leone. The relatively low frequency of the G119S mutation (0.14; N = 26) was consistent with the low phenotypic resistance to bendiocarb and full susceptibility to pirimiphos-methyl observed in this assessment. The absence of the G119S mutation in An. coluzzii cannot be explained by geographic variation, as both species were sympatric and collected from the same breeding sites. This finding is different from previous reports from West Africa, which observed this mutation in higher frequencies in both species: 0.32 in An. gambiae s.s. and 0.04 in An. coluzzii from Burkina Faso [ 40 ], 0.31 in An. gambiae s.s. and 0.35 in An. coluzzii from Côte d’Ivoire [ 42 , 43 ], and 0.24 in An. gambiae s.s. and 0.04 in An. coluzzii from Ghana [ 39 ]. The G119S mutation has been associated with a high fitness cost in Culex pipiens populations [ 37 , 44 ]. Indications of a high fitness cost were also similar in An. gambiae s.s. as the frequency of the Ace-1 mutation in mosquito populations was observed to decline rapidly after a few generations without selection pressure from organophosphates or carbamates [ 40 , 45 ]. In Burkina Faso, an excess of heterozygous genotypes in S form populations likely indicated that a fitness cost is associated with the mutation when present in a homozygous form. However, the findings from this assessment were different, such that all the Ace-1 G119S mutations were detected in the homozygous form with complete deficiency of heterozygotes and in significant deviation from the Hardy-Weinberg equilibrium. Nevertheless, the phenomenon of heterozygote deficiency in the G119S mutation is not new, as other studies have reported such a deficit in some sites in Cameroon [ 41 ] but does suggest a need for further exploration. In the study by Elanga-Ndille et al. [ 41 ] in Cameroon, each sequenced individual specimen possessed at least two distinct Ace-1 resistant alleles and one susceptible allele. This was a possible explanation for why most mosquitoes that remained alive after carbamate exposure were genotyped as homozygote resistant with a lack of heterozygotes: mosquitoes with two copies of the gene seemed to have three resistant alleles vs. only one susceptible allele. It is hypothesized that this duplication decreases the fitness cost associated with the resistant genotype [ 45 , 46 ] thereby hindering carbamate-based vector control strategies [ 35 ]. Since no mosquitoes survived the pirimiphos-methyl bioassays and the few that survived the bendiocarb exposure were not tested separately, we were unable to investigate the link between the G119S mutation and phenotypic resistance to carbamates and organophosphates. However, six of the 13 mosquitoes from the bioassay with bendiocarb were carrying the Ace -1 G119S mutation homozygous allele. The Ace-1 G119S mutation (associated with a low level of resistance to carbamate insecticides) was also present in An. gambiae s.s. but not in An. coluzzii . The G119S Ace-1 mutation was not associated with pirimiphos-methyl resistance. However, further bioassays and screening for the Ace-1 resistance allele on a wider scale would be required to understand the implications of the current status of the Ace-1 mutation for the efficacy of organophosphate insecticide use in vector control in Sierra Leone. Conclusion The resistance profile of An. gambiae s.l.in Sierra Leone is relatively similar to that of other West African countries. It is characterized by a high level of pyrethroid resistance and an almost fixed L1014F mutation coupled with evolving L1014S, G119S and N1575Y mutations. Sierra Leone should continue to monitor the effects of the 2020 distribution of PBO nets on insecticide resistance evolution in mosquito vectors and the resulting epidemiological impacts associated with malaria burden reduction. Additionally, given the high frequency of the L1014F mutation, the emergence of the N1575 mutation and the modest increase in the susceptibility of the vector after pre-exposure to PBO shown in this assessment, it would be prudent for the NMCP to maximize the use of nets treated with non-pyrethroids for future mass distribution cycles and routine distribution channels. This assessment has demonstrated that neonicotinoid- and pyrrole-based insecticides show promising results in terms of vector susceptibility, but further assessment of their residual bio-efficacy and other implementation-related factors such as cost, and community acceptance is required to select insecticides for evidence-based vector control. Taken as a whole, this assessment has generated important information on the resistance profile of the main malaria vectors against the most common insecticides used in Sierra Leone. These results have formed the basis for further entomological studies and supported the NMCP of Sierra Leone in its development of insecticide resistance monitoring and management strategies. Abbreviations ANC: Antenatal Care Ace-1 : Acetylcholinesterase 1 CDC: Centers for Disease Control and Prevention DDT : Dichlorodiphenyltrichloroethane DNA: Deoxyribonucleic Acid EPI: Expanded Program on Immunization IRS: Indoor Residual Spraying ITN: Insecticide-Treated Nets kdr: knock-down resistance NMCP: National Malaria Control Program PBO: Piperonyl Butoxide PCR: Polymerase Chain Reaction PMI: US President’s Malaria Initiative RFLP: Restriction Fragment Length Polymorphism VGSC Voltage-gated sodium channel WHO: World Health Organization Declarations Ethics approval and consent to participate. Not applicable. Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by the United States President’s Malaria Initiative through the United States Agency for International Development VectorLink Project. The opinions expressed herein are those of the authors and do not necessarily reflect the views of USAID, CDC, or PMI. Author’s Contributions EA was involved in the design of the study, provided the overall coordination of the assessment, performed, and supervised the tests, collection, and interpretation of data. KO supported the analysis, reviewed, and formatted the manuscript. YY was involved in data analysis and drafted the manuscript. RL, JD, TH, DS, RJ, and MM were involved in the design of the assessment, provided technical support, reviewed data, and edited the manuscript. SS and FY provided technical and management support. LI reviewed the manuscript and provided management support. All authors read and approved the final manuscript. Acknowledgements The technicians who conducted mosquito collections and insecticide resistance tests are thanked for their dedicated work. We thank the Center for Research in Infectious Diseases in Cameroon for performing the molecular analysis. We also would like to thank Linda Moll for her support in editing the draft. This paper is dedicated to the memory of our dear colleague and friend, NMCP Director Dr. Samuel Juana Smith , who passed away unexpectedly while this paper was in preparation. His contributions to fighting malaria in Sierra Leone, and thus to the successful implementation of the work contained herein, were immeasurable. References WHO: World malaria report 2022. 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20:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4720552/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4720552/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12936-025-05267-z","type":"published","date":"2025-03-13T15:58:06+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61769291,"identity":"ce037d91-9786-4837-b96d-465ab36458db","added_by":"auto","created_at":"2024-08-05 11:06:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1318700,"visible":true,"origin":"","legend":"\u003cp\u003eInsecticide and entomological monitoring sites in Sierra Leone\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4720552/v1/44cf6479d874086f95d955d9.png"},{"id":61768840,"identity":"a9a176d4-a064-4f05-8f0e-ffc7a20ff62f","added_by":"auto","created_at":"2024-08-05 10:58:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":258598,"visible":true,"origin":"","legend":"\u003cp\u003eSusceptibility status of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. to different concentrations (1x, 5x, and 10x) of pyrethroids and diagnostic doses of pirimiphos-methyl (0.25%) and bendiocarb (0.1%).\u003c/p\u003e\n\u003cp\u003eNote: Numbers on top of bars are total numbers of mosquitoes tested.\u003c/p\u003e\n\u003cp\u003e*Tests not done due to the insufficient numbers of mosquitoes collected\u003cem\u003e.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4720552/v1/b83c391a3b811e3fe255c63f.png"},{"id":61768838,"identity":"be856ea7-61f7-4e92-9340-1b4d912d4966","added_by":"auto","created_at":"2024-08-05 10:58:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58081,"visible":true,"origin":"","legend":"\u003cp\u003eMortality of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. to chlorfenapyr (100 µg/bottle) and clothianidin (2%).\u003c/p\u003e\n\u003cp\u003eNote: Numbers on top of bars are total number of mosquitoes tested\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4720552/v1/721db118ac784e0766641fd6.png"},{"id":61769290,"identity":"f0d99fbc-9b2d-496e-b175-3c591585b769","added_by":"auto","created_at":"2024-08-05 11:06:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":117812,"visible":true,"origin":"","legend":"\u003cp\u003eMortality of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. to alpha-cypermethrin (0.05%), deltamethrin (0.05%) and permethrin (0.75%) without and post-PBO exposure.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e*Numbers on top of bars are total numbers of mosquitoes tested.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4720552/v1/265881e1902f93adeaa76184.png"},{"id":78689306,"identity":"7936f94d-7ddc-4acf-ba2b-092856313b27","added_by":"auto","created_at":"2025-03-17 16:12:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3743809,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4720552/v1/46de3375-e9e9-4f91-9dac-ad08f2336b81.pdf"},{"id":61768837,"identity":"a993147e-838e-42e1-b4c6-768bd36ed65f","added_by":"auto","created_at":"2024-08-05 10:58:07","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":47053,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4720552/v1/45b0e108b9a8355726e7fb3e.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Profile and Characterization of Insecticide Resistance Mechanisms in the Anopheles gambiae s.l. Population of Sierra Leone","fulltext":[{"header":"Background","content":"\u003cp\u003eGlobally, an estimated 247\u0026nbsp;million cases of malaria and 619,000 deaths occurred in 2021 with 95% of the cases reported from Africa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The majority (96%) of malaria deaths occurred in Africa with children under five years of age accounting for 80% of the malaria deaths [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Between 2000 and 2015, global malaria case incidence declined by 27%, and by less than 2% between 2015 and 2019, and stagnated since 2021 indicating a slowing of the rate of decline since 2015 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Indoor residual spraying (IRS) and insecticide treated nets (ITNs) are considered core interventions in the global control of malaria, which have contributed to the decline in malaria cases globally over the last two decades [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the intensive use of insecticides in agriculture and to a lesser degree in public health has led to widespread resistance among targeted mosquito vectors [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and the major gains in the control of malaria have been threatened due to insecticide resistance [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMalaria is endemic in Sierra Leone, with stable and perennial transmission throughout the country. It is the leading cause of morbidity and mortality and accounts for 40.3% of all-age outpatient morbidity, 37.6% of hospitalizations, and has a case fatality rate of 17.6% in children under five years of age [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The Sierra Leone National Malaria Control Program (NMCP) conducted mass distribution of ITNs in 2010, 2014, 2017 and 2020 to achieve universal coverage. The mass distribution in May 2020 was conducted solely with ITNs impregnated with a pyrethroid and the synergist piperonyl-butoxide (PBO ITNs), making Sierra Leone the world\u0026rsquo;s first country to deploy PBO ITNs nationwide. The next mass distribution campaign using PBO ITNs and Interceptor G2 (IG2, active ingredients of chlorfenapyr and alpha-cypermethrin) ITNs is planned to begin in January 2024. The mass campaigns are complemented by routine IG2 or PBO ITN distribution through antenatal care (ANC) and Expanded Program on Immunization (EPI) clinics including during Maternal and Child Health Week. A pilot IRS campaign with lambda-cyhalothrin was implemented in four districts in 2011 and 2012, covering 5.8% of the population in Sierra Leone. In 2021, the NMCP in collaboration with the U.S. President\u0026rsquo;s Malaria Initiative (PMI) began implementing IRS with SumiShield\u0026trade; (active ingredient, clothianidin) in Bo and Bombali districts that had the highest entomological inoculation rates (EIR) of over 410 infectious bites/person/year with malaria prevalence of between 38 and 40% in children under 5 years [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSuccessful implementation of a vector control program relies on knowledge of vector species, behavior, infectivity, and susceptibility to insecticides [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Members of the \u003cem\u003eAnopheles gambiae\u003c/em\u003e complex and \u003cem\u003eAn. funestus\u003c/em\u003e group are the major vectors of malaria in sub-Saharan Africa (SSA), including Sierra Leone [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Prior to this study, \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. and \u003cem\u003eAn. coluzzii\u003c/em\u003e had been reported in Sierra Leone but their insecticide resistance status was not well established due to the disruption of entomological studies during the civil war (from 1991 to 2002) and beyond [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Characterization of insecticide resistance in mosquitoes was later reported, but only in urban Freetown [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The knock-down resistance \u003cem\u003e(kdr)\u003c/em\u003e mutation conferring resistance to pyrethroids and Dichlorodiphenyltrichloroethane (DDT), first reported in 1991 in \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. populations in C\u0026ocirc;te d\u0026rsquo;Ivoire [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], has spread within the species complex within SSA, including to Sierra Leonen[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Although some studies have indicated that ITNs remain effective for providing protection in areas with pyrethroid resistant vectors [\u003cspan additionalcitationids=\"CR17 CR18\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], other studies suggest that in areas where there is high levels of \u003cem\u003ekdr\u003c/em\u003e in local vector populations, pyrethroid treated ITNs or pyrethroid-based IRS products failed to control the \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. population [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The new generation ITNs incorporating PBO or dual active ingredients have been shown to be more effective against pyrethroid resistant mosquitoes [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo better guide deployment of vector control interventions in Sierra Leone, the NMCP in collaboration with PMI VectorLink project assessed the susceptibility of malaria vectors to common insecticides used in malaria control as well as the distribution of molecular markers of insecticide resistance between 2018\u0026ndash;19. We investigated the status of phenotypic resistance to insecticides in the pyrethroid, organophosphate, carbamate, neonicotinoid, and pyrrole classes of insecticides and determined the occurrence and frequency of molecular mechanisms for pyrethroid, organophosphate and carbamate resistance. The aims were to characterize resistance of the \u003cem\u003eAn. gambiae\u003c/em\u003e complex mosquitoes to insecticides currently in use and under consideration for use in IRS and ITNs, to inform decision making for optimizing vector control.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSampling area\u003c/h2\u003e \u003cp\u003eSierra Leone is located on the west coast of Africa, bordered on the north and east by Guinea, on the south by Liberia, and opens into the Atlantic Ocean to the West. The study was conducted in four sites in four districts (Bo, Bombali, Kono and Western Rural Area) representing the different geographical regions of Sierra Leone. Bo represents the southern region; Bombali the northern; Kono the eastern; and Western Area Rural the west. All the districts lie within the rainforest belt vegetation with Western Area Rural having a coastal ecosystem with brackish water due to the presence of Atlantic Ocean. The rainfall pattern is similar in all sites, beginning in May and ending in October. The dry season begins in November through to April. In each district, mosquitoes were collected from one rural chiefdom: Jaima Bongor in Bo, Gbanti Kamaranka in Bombali, Nimyama in Kono and Koya in Western Area Rural (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The four sites are also where the PMI VectorLink project in Sierra Leone has been doing comprehensive entomological monitoring activities since 2018.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eInsecticide susceptibility tests\u003c/h2\u003e \u003cp\u003eWorld Health Organization (WHO) tube tests [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and Centers for Disease Control and Prevention (CDC) bottle bioassays [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] were performed to assess the susceptibility of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. to the most common insecticides used in ITNs and IRS in Sierra Leone. The WHO impregnated test papers at diagnostic doses (1x) of pyrethroids (alpha-cypermethrin (0.05%), deltamethrin (0.05%) and permethrin (0.75%), pirimiphos methyl (0.25%) and bendiocarb (0.1%) were used to assess mosquito susceptibility. The WHO tube method was used to test \u003cem\u003eAn. gambiae\u003c/em\u003e s.l., susceptibility to pirimiphos methyl (0.25%) in all four sites and to bendiocarb (0.1%) in all sites except in Bombali, where insufficient mosquitoes were collected. The CDC bottle assay was used to assess the intensity of resistance to pyrethroids.\u003c/p\u003e \u003cp\u003eLarvae and pupae of \u003cem\u003eAnopheles\u003c/em\u003e mosquitoes were collected from breeding sites, mainly from temporary rain pools, in and around the sentinel sites and reared to adults at the Vector-Borne Disease Insectary and Laboratory (VBDIL) in Makeni, Bombali District. Mosquitoes were morphologically identified at the adult stage and only \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. aged between 2\u0026ndash;5 days old were used for the susceptibility tests. Tests were conducted in all four districts: Bo, Bombali, Kono and Western Rural Area. The number of dead and alive mosquitoes in both the exposure and the control tubes were recorded after a 24-hour post-exposure holding period. Given the slow-acting nature of clothianidin, post-exposure mortalities were scored every 24 hours for up to five days or until 100% mortality was recorded, whichever came first.\u003c/p\u003e \u003cp\u003eThe CDC bottle bioassay was also used to assess susceptibility of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. to chlorfenapyr. Two-to-five-day old \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. reared from larvae were exposed to 250ml Wheaton bottles treated with a diagnostic concentration of 100\u0026micro;g/bottle chlorfenapyr. For clothianidin, the bottles were coated with 2% clothianidin. Tests with \u003cem\u003eAn. gambiae\u003c/em\u003e Kisumu as positive controls and negative controls without insecticide were run in parallel. Female \u003cem\u003eAnophele\u003c/em\u003es mosquitoes were introduced in batches of 20\u0026ndash;25 in each replicate. After a 60 minutes exposure period, mosquitoes were released into clean cages and then gently aspirated into labeled paper cups covered with untreated netting and provided with 10% sugar solution. Knock-down was recorded 60 minutes after the start of the test, while mosquitoes were still in the bottle. Mortality was recorded every 24 hours for up to five days or until 100% mortality was recorded, whichever came first.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSynergist, and intensity assays of pyrethroids\u003c/h2\u003e \u003cp\u003eFirst, mosquitoes were exposed to treated WHO papers at diagnostic doses (1x) of pyrethroids (alpha-cypermethrin (0.05%), deltamethrin (0.05%) and permethrin (0.75%)) with and without piperonyl butoxide 4% (PBO) synergist using the WHO procedure [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Adult \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. mosquitoes aged 2\u0026ndash;5 days old were pre-exposed to PBO for an hour followed by exposure to either deltamethrin, permethrin and alpha-cypermethrin impregnated papers (1x) in WHO tube tests. The number of dead and alive mosquitoes in both the exposure and the control tubes were recorded after a 24-hour post-exposure holding period. The mortality estimates in the insecticide-only group, without pre-exposure to PBO, at diagnostic doses were also taken as 1x in the intensity assays.\u003c/p\u003e \u003cp\u003eThe CDC bottle bioassay [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] was used to assess the intensity of resistance to pyrethroids above the diagnostic dose. \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. adult mosquitoes were exposed to alpha-cypermethrin and deltamethrin at 62.5 (5x) and 125 (10x) \u0026micro;g/bottle. Permethrin was tested at a dose of 107.5 (5x) and 215 (10x) \u0026micro;g/bottle. These intensities were estimated in all sites except Kono, where the number of larvae collected was only enough to measure the intensity of alpha-cypermethrin resistance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMolecular species identification\u003c/h2\u003e \u003cp\u003eA subset of 200 mosquitoes out of 5,326, composed of those that survived or died following insecticide exposures, and representing all the districts that were tested were randomly sampled for molecular analysis. Genomic DNA was extracted from each mosquito sample via the Livak method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and stored at -20\u0026ordm;C following extraction. Identification of members of the \u003cem\u003eAn. gambiae\u003c/em\u003e complex was done using SINE polymerase chain reaction (PCR) that allows identification of \u003cem\u003eAn. gambiae\u003c/em\u003e s.s., \u003cem\u003eAn. coluzzii\u003c/em\u003e and \u003cem\u003eAn. arabiensis\u003c/em\u003e [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In coastal sites where other species such as \u003cem\u003eAn. melas\u003c/em\u003e were suspected, the PCR-restriction fragment length polymorphism (PCR-RFLP) protocol described by Scott et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] was used. PCR products were run via electrophoresis through a 1.5% agarose gel with Midori Green\u0026reg; (Gene flow, UK) and visualized using ultraviolet light.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMolecular characterization of resistance mechanisms\u003c/h2\u003e \u003cp\u003eThe subset samples above were also screened for the presence of the 1014F, 1014S, N1575Y, Ace-1G119S mutations using TaqMan assays [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Genotypes were scored from scatter plots of results produced by the Mx3005 v4.10 software. Three positive samples of known genotypes for each of the alleles were used as positive controls while distilled water was used as negative control for each of the experiments as described in the protocols ([\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eMortality was calculated by dividing the number of dead mosquitoes following exposure by total number exposed for each insecticide. Mortalities were corrected using Abbott\u0026rsquo;s formula if the mortality of control mosquitoes exposed to solvent only was \u0026ge;\u0026thinsp;5% and \u0026lt;\u0026thinsp;20%. Tests were discarded and repeated if control mortalities were \u0026ge;\u0026thinsp;20% [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Test results were also discarded and repeated if the mortality of mosquitoes exposed to PBO only was \u0026gt;\u0026thinsp;10%.\u003c/p\u003e \u003cp\u003eSusceptibility levels of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. were evaluated based on the WHO criteria of test mortality [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]: corrected mortality of 98\u0026ndash;100% after 24\u0026ndash;120 hours post exposure indicated susceptibility; corrected mortality, \u0026gt; 90% but \u0026lt;\u0026thinsp;98%, indicated the existence of possible resistance; and mortality of \u0026lt;\u0026thinsp;90% indicated the presence of resistant individuals in the vector population.\u003c/p\u003e \u003cp\u003eGenotype frequencies per species per site was calculated as the relative frequency of the homozygote resistant and heterozygote resistant individuals. The allelic frequencies of L1014F, L1014S, N1575Y and \u003cem\u003eAce-1\u003c/em\u003e were calculated as follows: F(R) = [2RR\u0026thinsp;+\u0026thinsp;RS]/ [2(RR\u0026thinsp;+\u0026thinsp;RS\u0026thinsp;+\u0026thinsp;SS)]. The Hardy-Weinberg equation was used to calculate the expected genotype frequency of L1014F, L1014S, N1575Y and \u003cem\u003eAce-1\u003c/em\u003e in \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. and \u003cem\u003eAn. coluzzii.\u003c/em\u003e The expected and observed genotype frequencies were compared using Pearson\u0026rsquo;s Chi-squared tests in Microsoft Excel 2016 to determine statistical significance of differences and estimate inbreeding co-efficient (F\u003csub\u003eIS\u003c/sub\u003e) and STATA-SE12 was used to generate confidence intervals for the resistance allelic frequency distributions.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\"\u003e\n \u003ch2\u003eInsecticide susceptibility and intensity assays\u003c/h2\u003e\n \u003cp\u003eA total of 5,326 \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. were successfully reared to adults and exposed to different insecticides (supplementary material Table\u0026nbsp;1). \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. from all sampling sites were resistant to the diagnostic dose of deltamethrin, permethrin and alpha-cypermethrin (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e, supplementary material Table\u0026nbsp;1). The 24-hour post-exposure mortality ranged from 7% in Bombali to 65% in Western Area Rural for permethrin; from 12% in Bombali to 53% in Kono for deltamethrin; and from 10% in Bo to 50% in Western Area Rural for alpha-cypermethrin (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e). \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. was fully susceptible to pirimiphos-methyl in all sites. For bendiocarb, there was resistance in Western Area Rural with 88% mortality and possible resistance in Bo and Kono with 94% mortality. Resistance intensity to alpha-cypermethrin, deltamethrin and permethrin was high at all sites, with mortality rates of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. below 98% at 10 times (10x) the diagnostic dose (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e). Mortality was below 90% at five times (5x) the diagnostic dose for all the three pyrethroids except for alpha-cypermethrin in Bombali (Fig.\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. was susceptible to chlorfenapyr in all four sampling sites (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e). In all sites, over 98% of mosquitoes exposed to chlorfenapyr died after 72-hours post-exposure with mortality reaching 100% after 120 hours (4 days post exposure) (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e). \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. was also fully susceptible to clothianidin at all sites (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e). In Western Rural Area and Bombali, 100% mortality to clothianidin was recorded within the 24-hour holding period. However, it took 72 hours to achieve 100% mortality in Bo and 120 hours in Kono (Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e). Overall, mosquitoes died faster after exposure to clothianidin than to chlorfenapyr. At 24 hours holding time, the mean percent mortality was significantly higher (ꭓ\u003csup\u003e2\u003c/sup\u003e = 272.5; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) for clothianidin (94.1%) than for chlorfenapyr (35.9%) (Table\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e). The difference narrowed at 48 hours holding time, but it was still significantly higher (ꭓ\u003csup\u003e2\u003c/sup\u003e = 4.8; p\u0026thinsp;=\u0026thinsp;0.028) for clothianidin than for chlorfenapyr. There was no difference at 72 hours holding time (ꭓ\u003csup\u003e2\u003c/sup\u003e = 1.2; p\u0026thinsp;=\u0026thinsp;0.273) and beyond (Table\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eMean percent mortality of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. by holding time post exposure to chlorfenapyr and clothianidin.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eHolding time\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean mortality\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e24hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e48hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e72hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e96hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e120hrs\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChlorfenapyr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e95.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eClothianidin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTest\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eꭓ\u003csup\u003e2\u003c/sup\u003e = 272.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eꭓ\u003csup\u003e2\u003c/sup\u003e = 4.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eꭓ\u003csup\u003e2\u003c/sup\u003e = 0.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eꭓ\u003csup\u003e2\u003c/sup\u003e = 0.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eP value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.028\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.644\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eSynergist assays\u003c/h2\u003e\n \u003cp\u003eExposure to 4% PBO increased mortality for each pyrethroid insecticide but full restoration of susceptibility above 98% was not achieved for any insecticides in any locations. The absolute increase after pre-exposure for deltamethrin ranged from 43.9% in Bombali to 52.9% in Bo. For alpha-cypermethrin, the absolute increase ranged between 41.7% in Bo and 56.0% in Kono. The absolute increase for permethrin was highest in Kono (66.3%) and lowest in Western Rural Area (41.5%) (Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eMolecular species identification\u003c/h2\u003e\n \u003cp\u003eA total of 191 out of 200 (95.5%) mosquitoes were successfully identified to sibling species. \u003cem\u003eAnopheles gambiae\u003c/em\u003e was dominant (81.7% 95% CI (75.3\u0026ndash;86.7), followed by \u003cem\u003eAn. coluzzii\u003c/em\u003e (17.8%, 95% CI (12.8\u0026ndash;24.1). One specimen was identified as a hybrid of \u003cem\u003eAn. gambiae and An. coluzzii\u003c/em\u003e 0.5%, 95% CI (0.03\u0026ndash;3.3).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eMolecular characterization of resistance mechanisms\u003c/h2\u003e\n \u003cp\u003eThe frequency of the \u003cem\u003ekdr\u003c/em\u003e L1014F resistance (r) allele was high, 0.98 (373/380); 95% CI: 0.94\u0026ndash;0.99) whereas the \u003cem\u003ekdr\u003c/em\u003e L1014S mutation frequency was low at 0.02 (8/382); 95% CI: 0.01\u0026ndash;0.04) across all four assessment sites (Table\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e). The wild-type allele for \u003cem\u003ekdr\u003c/em\u003e L1014F was not detected in any of the \u003cem\u003eAn. gambiae\u003c/em\u003e or \u003cem\u003eAn. coluzzii\u003c/em\u003e specimens screened (Tables\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e and \u003cspan\u003e3\u003c/span\u003e). The N1575Y mutation, which amplifies resistance conferred by L1014F and L1014S, was detected at a frequency of 0.089 (34/384) and were all in a heterozygous form (Tables\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e and \u003cspan\u003e3\u003c/span\u003e). The difference in the frequency of the N1575Y mutation between \u003cem\u003eAn. gambiae\u003c/em\u003e (0.095) and \u003cem\u003eAn. coluzzii\u003c/em\u003e (0.059) was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.928). All 34 samples with the N1575Y mutation also carried the homozygous mutant allele L1014F. No deviation from the Hardy-Weinberg equilibrium and no deficiency of heterozygous individuals was detected at the L1014F, L1014S and N1575Y loci (Tables\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e and \u003cspan\u003e4\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003cp\u003eTable\u0026nbsp;2: Distribution of molecular markers of insecticide resistance by phenotype in Sierra Leone\u003c/p\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"864\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.202312138728324%\" colspan=\"2\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"17.803468208092486%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eKdr-w\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.15028901734104%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eKdr-e\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.728323699421967%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u003cem\u003eAce-1\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.115606936416185%\" colspan=\"4\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eN1575Y\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.127020785219399%\" valign=\"top\"\u003e\n \u003cp\u003eDistrict\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.046189376443419%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePhenotype\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.348729792147806%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLF\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.351039260969977%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.618937644341801%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eLL\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSS\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.466512702078522%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNY\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eNA\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.127020785219399%\" valign=\"top\"\u003e\n \u003cp\u003eBo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.046189376443419%\" valign=\"top\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.348729792147806%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.351039260969977%\" valign=\"top\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.618937644341801%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.466512702078522%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.127020785219399%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.046189376443419%\" valign=\"top\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.348729792147806%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.351039260969977%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.618937644341801%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.466512702078522%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.127020785219399%\" valign=\"top\"\u003e\n \u003cp\u003eBombali\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.046189376443419%\" valign=\"top\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.348729792147806%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.351039260969977%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.618937644341801%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.466512702078522%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.127020785219399%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.046189376443419%\" valign=\"top\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.348729792147806%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.351039260969977%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.618937644341801%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.466512702078522%\" valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.127020785219399%\" valign=\"top\"\u003e\n \u003cp\u003eKono\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.046189376443419%\" valign=\"top\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.348729792147806%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.351039260969977%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.618937644341801%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.466512702078522%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.127020785219399%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.046189376443419%\" valign=\"top\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.348729792147806%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.351039260969977%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.618937644341801%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.466512702078522%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.127020785219399%\" valign=\"top\"\u003e\n \u003cp\u003eWestern Rural Area\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.046189376443419%\" valign=\"top\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.348729792147806%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.351039260969977%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.618937644341801%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.466512702078522%\" valign=\"top\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.127020785219399%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"10.046189376443419%\" valign=\"top\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.348729792147806%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"6.351039260969977%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.618937644341801%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.466512702078522%\" valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.127020785219399%\" valign=\"top\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.046189376443419%\" valign=\"top\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e191\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.348729792147806%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.351039260969977%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e201\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.618937644341801%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e9\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e188\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e201\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e173\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.311778290993072%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e201\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"6.466512702078522%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e35\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"4.272517321016166%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e164\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"3.810623556581986%\" valign=\"top\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"5.542725173210162%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e201\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eNA = Not amplified; LL = homozygous wild type; LF = heterozygous resistant for \u003cem\u003ekdr-w\u003c/em\u003e; FF = homozygous resistant for \u003cem\u003ekdr-w\u003c/em\u003e; LS= heterozygous resistant for \u003cem\u003ekdr-e\u003c/em\u003e; GG= homozygous wild type; SS = homozygous resistant for \u003cem\u003eAce\u003c/em\u003e-1; NN = homozygous wild type for N1575Y; NY = heterozygous resistant for N15756Y.\u003c/p\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eAllelic frequencies of L1014F and L1014S\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"16\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSite\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"14\"\u003e\n \u003cp\u003eL1014\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eFF\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ef (L1014F)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eF\u003csub\u003eIS\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLL\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ef(L1014S)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eF\u003csub\u003eIS\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eBo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. coluzzii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.85-1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.03030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.0303\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.91-1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.01695\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.01\u0026ndash;0.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.05263\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eBombali\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. coluzzii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.74-1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.27)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(96.0\u0026ndash;1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.01\u0026ndash;0.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.03529\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eKono (Eastern)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. coluzzii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.47-1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.14286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.53)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.14286\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.98\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.92-1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.02439\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.04)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eWestern Rural Area\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. coluzzii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.57\u0026ndash;0.98)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.16667\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(95.0\u0026ndash;1.00)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e190\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e183\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.98\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e(0.94\u0026ndash;0.99)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.01799\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e191\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e183\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e8\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.02\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e(0.01\u0026ndash;0.04)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.02139\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"16\"\u003eN = # tested; LL\u0026thinsp;=\u0026thinsp;homozygous wild type; LF\u0026thinsp;=\u0026thinsp;heterozygous resistant for \u003cem\u003ekdr-w\u003c/em\u003e; FF\u0026thinsp;=\u0026thinsp;homozygous resistant for \u003cem\u003ekdr-w\u003c/em\u003e; f (L1014F)\u0026thinsp;=\u0026thinsp;frequency of the L1014F resistant allele; LS\u0026thinsp;=\u0026thinsp;heterozygous resistant for \u003cem\u003ekdr-e\u003c/em\u003e; SS\u0026thinsp;=\u0026thinsp;homozygous resistant for \u003cem\u003ekdr-e\u003c/em\u003e; f (L1014S)\u0026thinsp;=\u0026thinsp;frequency of the L1014S resistant allele.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"16\"\u003eF\u003csub\u003eIS\u003c/sub\u003e Values\u0026thinsp;\u0026gt;\u0026thinsp;0 indicate heterozygote deficiency, while values\u0026thinsp;\u0026lt;\u0026thinsp;0 indicate heterozygote excess.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eAllelic frequencies of N1575Y and G119S\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"16\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSite\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"8\"\u003e\n \u003cp\u003eN1575Y\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"7\"\u003e\n \u003cp\u003eG119S\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNY\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eYY\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ef(N1575Y)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eF\u003csub\u003eIS\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGG\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSS\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ef(G119S)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e95% CI\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eF\u003csub\u003eIS\u003c/sub\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eBo\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. coluzzii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.03030\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.01\u0026ndash;0.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.05263\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.08\u0026ndash;0.29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eBombali\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. coluzzii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.02\u0026ndash;0.48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.20000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.26)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.06\u0026ndash;0.21)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.14286\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.03\u0026ndash;0.14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eKono\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. coluzzii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.02\u0026ndash;0.13)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.06024\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.06\u0026ndash;0.20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cstrong\u003eWestern Rural Area\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. coluzzii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.07692\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.00-0.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.07\u0026ndash;0.23)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.15942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e40\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(0.22\u0026ndash;0.44)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eTotal\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e192\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e158\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e34\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.09\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e(0.06\u0026ndash;0.12)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-0.09714\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e191\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e165\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e26\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.14\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e(0.01\u0026ndash;0.17)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.000\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"16\"\u003eN = # tested; GG\u0026thinsp;=\u0026thinsp;homozygous wild type; GS\u0026thinsp;=\u0026thinsp;heterozygous resistant for \u003cem\u003eAce\u003c/em\u003e-1; SS\u0026thinsp;=\u0026thinsp;homozygous resistant for \u003cem\u003eAce\u003c/em\u003e-1; f (G119S)\u0026thinsp;=\u0026thinsp;frequency of the G119S resistant allele; NN\u0026thinsp;=\u0026thinsp;homozypous wild type for N1575Y; NY\u0026thinsp;=\u0026thinsp;heterozygous resistant for N15756Y; YY\u0026thinsp;=\u0026thinsp;homozygous resistant for N1575Y; f (N1575Y)\u0026thinsp;=\u0026thinsp;frequency of the N1575Y resistant allele.\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"16\"\u003eF\u003csub\u003eIS\u003c/sub\u003e values\u0026thinsp;\u0026gt;\u0026thinsp;0 indicate heterozygote deficiency, while values\u0026thinsp;\u0026lt;\u0026thinsp;0 indicate heterozygote excess.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe overall frequency of the G119S mutation was 0.14 (N\u0026thinsp;=\u0026thinsp;52). No G119S mutation was found in \u003cem\u003eAn. coluzzii\u003c/em\u003e (Table\u0026nbsp;\u003cspan\u003e4\u003c/span\u003e). However, both deviation from the Hardy-Weinberg equilibrium and deficiency of heterozygotes was detected at the G119S locus of \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. and was statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Table\u0026nbsp;\u003cspan\u003e5\u003c/span\u003e). As all the samples were either homozygous or heterozygous to L1014F mutation, all the specimens with L1014S, G119S and N1575Y also had the L1014F resistance allele. All mosquitoes with L1014S did not have the G119S mutation and only one was carrying the N1575Y mutation. Seven \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. specimens were found to be carrying three mutations: L1014F plus either L1014S or G119S, or L1014F plus G119S and N1575Y (Table\u0026nbsp;\u003cspan\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 5\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eGenotype frequencies for the L1014 kdr, G119S, and N1575Y mutation in \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. from Sierra Leone. Tests of Hardy-Weinberg Equilibrium (\u0026chi;2) with corresponding P-values.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMutation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSite\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eObserved\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u0026chi;2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eP-VALUE\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRR\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eL1014F\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBo (Southern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.960\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8853\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBombali (Northern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWestern Rural Area (Western)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.960\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.040\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0208\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8853\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKono (Eastern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.938\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.063\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0499\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8232\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eL1014S\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBo (Southern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.898\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1416\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7067\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBombali (Northern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.060\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.940\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0478\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.8269\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWestern Rural Area (Western)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKono (Eastern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.020\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.980\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0052\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9425\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eG119S\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBo (Southern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;.00001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBombali (Northern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.060\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.940\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;.00001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWestern Rural Area (Western)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.740\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;.00001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKono (Eastern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.102\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.898\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49.0000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;\u0026thinsp;.00001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003e\u003cstrong\u003eN1575Y\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBo (Southern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1385\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7098\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBombali (Northern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.740\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.1164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2907\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eWestern Rural Area (Western)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.245\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.755\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9540\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.3287\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eKono (Eastern)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.000\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.900\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1385\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7098\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab7\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 6\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eMultiple mutations in \u003cem\u003eAn. gambiae\u003c/em\u003e s.l.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"9\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMutations\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eL1014F\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eL1014S\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eG119S\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eN1575Y\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePhenotype\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eResistant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSusceptible\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eL1014F\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eL1014S\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eG119S\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eN1575Y\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e--\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"9\"\u003e\u003cem\u003e*Also carry the L1014F mutation; 7 samples were with 3 types of mutations\u003c/em\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eSeven of the eight samples with the G119S mutation were also survivors of the exposure to bendiocarb. However, 68.2% (15/22) of the samples that were phenotypically resistant to bendiocarb were not carrying the G119S mutation. Ninety-five percent (19/20) of the samples susceptible to bendiocarb and pirimiphos-methyl were also not carrying the mutation (Table\u0026nbsp;\u003cspan\u003e7\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab8\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 7\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003ePhenotypic resistance of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. to bendiocarb and allelic frequencies of G119S\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eSpecies\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePhenotype\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e\u003cem\u003eG119S\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGG\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eGS\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eSS\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eOdds ratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eAn. gambiae\u003c/em\u003e s.l.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eResistant/Alive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.7, p\u0026thinsp;=\u0026thinsp;0.03\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSusceptible/Dead\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRef\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eTotal\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eGG\u0026thinsp;=\u0026thinsp;homozygous wild type; GS\u0026thinsp;=\u0026thinsp;heterozygous resistant for \u003cem\u003eAce\u003c/em\u003e-1; SS\u0026thinsp;=\u0026thinsp;homozygous resistant for \u003cem\u003eAce\u003c/em\u003e-1\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.s. was the dominant malaria vector in Sierra Leone followed by \u003cem\u003eAn. coluzzii\u003c/em\u003e. Consistent with previous studies, they were the only species sampled in larval habitats [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The main malaria vector in Sierra Leone, \u003cem\u003eAn. gambiae\u003c/em\u003e s.l., was highly resistant to the three pyrethroid insecticides tested with partial restoration of susceptibility following pre-exposure to PBO. The presence of survivors following exposure to 10x the discriminating doses is indicative of the high intensity of resistance to pyrethroids. The observed range of enhancement of mortality due to pre-exposure to PBO was similar across the three pyrethroids tested.\u003c/p\u003e \u003cp\u003eThis assessment also demonstrated the \u003cem\u003ekdr\u003c/em\u003e L1014F resistance mutation to be present at high frequency, which is not surprising, considering the high level of phenotypic resistance to pyrethroids. Though not frequently identified in previous samples, this report is a first of \u003cem\u003ekdr\u003c/em\u003e L1014S and N1575Y mutations in Sierra Leone. The N1575Y mutation, located in the \u003cem\u003eVgsc\u003c/em\u003e and believed to have a synergistic effect on pyrethroid and DDT resistance when combined with the L1014F mutation was previously reported from Burkina Faso, Benin, Cameroon and C\u0026ocirc;te d\u0026rsquo;Ivoire [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe detection of the N1575Y mutation in Sierra Leone requires more investigation to better characterize its expected synergistic relationship with 1014F \u003cem\u003ekdr\u003c/em\u003e. The presence of this additional mechanism that could further reduce insecticide efficacy in the already pyrethroid-resistant mosquitoes in Sierra Leone is concerning. Thus, monitoring of N1575Y should continue in order to understand its contribution to insecticide resistance among local vectors. This resistance mechanism could spread very rapidly [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and threaten the malaria vector control efforts in Sierra Leone which had relied primarily on the distribution of pyrethroid-treated ITNs. This finding also provides evidence for justifying the need to move to the distribution of non-pyrethroid nets.\u003c/p\u003e \u003cp\u003eAs the effect of PBO exposure was similar across the three pyrethroids tested, nets treated with any of the pyrethroids plus PBO might have similar effects and can be used as alternative tools in the national Sierra Leone Insecticide Resistance Management Plan. Indeed, NMCP distributed PermaNet 3.0 (deltamethrin\u0026thinsp;+\u0026thinsp;PBO) and Olyset Plus (permethrin\u0026thinsp;+\u0026thinsp;PBO) in the 2020 mass net campaign and these contributed to malaria reduction in Sierra Leone [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. These data not only yielded the evidence that PBO nets could provide better protection than pyrethroid nets without PBO in Sierra Leone, but also demonstrated that in the absence of non-pyrethroid ITNs available at the time of this assessment, Sierra Leone had the flexibility to choose PBO ITNs treated with any of the three pyrethroids. Sierra Leone was the world\u0026rsquo;s first country to provide population-level coverage nationally with next-generation PBO ITNs during a mass distribution campaign and evaluations to measure their durability and performance are underway. The NMCP has now incorporated these data and subsequent data on insecticide resistance to procure IG2 nets for 2023 mass ITN campaign.\u003c/p\u003e \u003cp\u003e \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. was susceptible to pirimiphos-methyl (organophosphate), chlorfenapyr (pyrrole) and clothianidin (neonicotinoid). However, there was an indication of possible resistance to bendiocarb (carbamate). As the NMCP prepared to implement IRS in Bo and Bombali districts in May/June 2021 at the start of the rainy season, the findings from this assessment provided critical data that guided the selection of an appropriate insecticide for the IRS program. Thus, clothianidin was selected for IRS because vectors were fully susceptible to it. There was no phenotypic resistance to pirimiphos-methyl and low-level resistance to bendiocarb was reported. However, carbamates and organophosphates are known to share the \u003cem\u003eAce\u003c/em\u003e-\u003cem\u003e1\u003c/em\u003e pathway as a resistance mechanism [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. With the reported phenotypic resistance to bendiocarb and the reported prevalence of the G119S mutation at 13.6% in the vector population, this mutation needs close monitoring to guide future IRS in Sierra Leone. Moreover, the combination of IRS using pirimiphos-methyl and PBO nets have been suggested to be antagonistic [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Thus, with the mass distribution of PBO nets that occurred in Sierra Leone in 2020 and continued PBO ITN distribution through some routine channels, and considering this potential antagonism between vector control interventions, caution should be used in the selection of pirimiphos-methyl for IRS. The susceptibility of the vector to chlorfenapyr also suggests that its use for IRS or as part of ITN treatment is a possibility when it becomes available.\u003c/p\u003e \u003cp\u003eThe presence of three or more \u003cem\u003eAce-1\u003c/em\u003e alleles in \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. mosquitoes was previously documented in several countries in West Africa [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, prior to the present assessment, little information was available for Sierra Leone. Although the G119S mutation is indicative of the phenotypic resistance to bendiocarb in this study, the high number of wild types that survived exposure to bendiocarb indicates involvement of other resistance mechanisms in Sierra Leone. The relatively low frequency of the G119S mutation (0.14; N\u0026thinsp;=\u0026thinsp;26) was consistent with the low phenotypic resistance to bendiocarb and full susceptibility to pirimiphos-methyl observed in this assessment.\u003c/p\u003e \u003cp\u003eThe absence of the G119S mutation in \u003cem\u003eAn. coluzzii\u003c/em\u003e cannot be explained by geographic variation, as both species were sympatric and collected from the same breeding sites. This finding is different from previous reports from West Africa, which observed this mutation in higher frequencies in both species: 0.32 in \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. and 0.04 in \u003cem\u003eAn. coluzzii\u003c/em\u003e from Burkina Faso [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], 0.31 in \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. and 0.35 in \u003cem\u003eAn. coluzzii\u003c/em\u003e from C\u0026ocirc;te d\u0026rsquo;Ivoire [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], and 0.24 in \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. and 0.04 in \u003cem\u003eAn. coluzzii\u003c/em\u003e from Ghana [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The G119S mutation has been associated with a high fitness cost in \u003cem\u003eCulex pipiens\u003c/em\u003e populations [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Indications of a high fitness cost were also similar in \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. as the frequency of the \u003cem\u003eAce-1\u003c/em\u003e mutation in mosquito populations was observed to decline rapidly after a few generations without selection pressure from organophosphates or carbamates [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In Burkina Faso, an excess of heterozygous genotypes in S form populations likely indicated that a fitness cost is associated with the mutation when present in a homozygous form. However, the findings from this assessment were different, such that all the \u003cem\u003eAce-1\u003c/em\u003e G119S mutations were detected in the homozygous form with complete deficiency of heterozygotes and in significant deviation from the Hardy-Weinberg equilibrium. Nevertheless, the phenomenon of heterozygote deficiency in the G119S mutation is not new, as other studies have reported such a deficit in some sites in Cameroon [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] but does suggest a need for further exploration.\u003c/p\u003e \u003cp\u003eIn the study by Elanga-Ndille et al. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] in Cameroon, each sequenced individual specimen possessed at least two distinct \u003cem\u003eAce-1\u003c/em\u003e resistant alleles and one susceptible allele. This was a possible explanation for why most mosquitoes that remained alive after carbamate exposure were genotyped as homozygote resistant with a lack of heterozygotes: mosquitoes with two copies of the gene seemed to have three resistant alleles vs. only one susceptible allele. It is hypothesized that this duplication decreases the fitness cost associated with the resistant genotype [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] thereby hindering carbamate-based vector control strategies [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Since no mosquitoes survived the pirimiphos-methyl bioassays and the few that survived the bendiocarb exposure were not tested separately, we were unable to investigate the link between the G119S mutation and phenotypic resistance to carbamates and organophosphates. However, six of the 13 mosquitoes from the bioassay with bendiocarb were carrying the \u003cem\u003eAce\u003c/em\u003e-1 G119S mutation homozygous allele.\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eAce-1\u003c/em\u003e G119S mutation (associated with a low level of resistance to carbamate insecticides) was also present in \u003cem\u003eAn. gambiae\u003c/em\u003e s.s. but not in \u003cem\u003eAn. coluzzii\u003c/em\u003e. The G119S \u003cem\u003eAce-1\u003c/em\u003e mutation was not associated with pirimiphos-methyl resistance. However, further bioassays and screening for the \u003cem\u003eAce-1\u003c/em\u003e resistance allele on a wider scale would be required to understand the implications of the current status of the \u003cem\u003eAce-1\u003c/em\u003e mutation for the efficacy of organophosphate insecticide use in vector control in Sierra Leone.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe resistance profile of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l.in Sierra Leone is relatively similar to that of other West African countries. It is characterized by a high level of pyrethroid resistance and an almost fixed L1014F mutation coupled with evolving L1014S, G119S and N1575Y mutations. Sierra Leone should continue to monitor the effects of the 2020 distribution of PBO nets on insecticide resistance evolution in mosquito vectors and the resulting epidemiological impacts associated with malaria burden reduction. Additionally, given the high frequency of the L1014F mutation, the emergence of the N1575 mutation and the modest increase in the susceptibility of the vector after pre-exposure to PBO shown in this assessment, it would be prudent for the NMCP to maximize the use of nets treated with non-pyrethroids for future mass distribution cycles and routine distribution channels. This assessment has demonstrated that neonicotinoid- and pyrrole-based insecticides show promising results in terms of vector susceptibility, but further assessment of their residual bio-efficacy and other implementation-related factors such as cost, and community acceptance is required to select insecticides for evidence-based vector control. Taken as a whole, this assessment has generated important information on the resistance profile of the main malaria vectors against the most common insecticides used in Sierra Leone. These results have formed the basis for further entomological studies and supported the NMCP of Sierra Leone in its development of insecticide resistance monitoring and management strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eANC:\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Antenatal Care\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAce-1\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e: \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eAcetylcholinesterase 1\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDC:\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Centers for Disease Control and Prevention\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDDT\u003c/strong\u003e:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Dichlorodiphenyltrichloroethane\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA:\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Deoxyribonucleic Acid\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEPI:\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Expanded Program on Immunization\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIRS:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eIndoor Residual Spraying\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eITN:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eInsecticide-Treated Nets\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003ekdr:\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/em\u003eknock-down resistance\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNMCP:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eNational Malaria Control Program\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePBO:\u003c/strong\u003e \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Piperonyl Butoxide\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR:\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Polymerase Chain Reaction\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePMI:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003eUS President\u0026rsquo;s Malaria Initiative\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRFLP:\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Restriction Fragment Length Polymorphism\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVGSC\u003c/strong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Voltage-gated\u0026nbsp;sodium channel\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWHO:\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003eWorld Health Organization\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the United States President\u0026rsquo;s Malaria Initiative through the United States Agency for International Development VectorLink Project. The opinions expressed herein are those of the authors and do not necessarily reflect the views of USAID, CDC, or PMI.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEA was involved in the design of the study, provided the overall coordination of the assessment, performed, and supervised the tests, collection, and interpretation of data. KO supported the analysis, reviewed, and formatted the manuscript. YY was involved in data analysis and drafted the manuscript. RL, JD, TH, DS, RJ, and MM were involved in the design of the assessment, provided technical support, reviewed data, and edited the manuscript. SS and FY provided technical and management support. LI reviewed the manuscript and provided management support. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe technicians who conducted mosquito collections and insecticide resistance tests are thanked for their dedicated work. We thank the\u0026nbsp;Center for Research in Infectious Diseases in Cameroon for performing the molecular analysis. We also would like to thank Linda Moll for her support in editing the draft.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThis paper is dedicated to the memory of our dear\u0026nbsp;\u003c/em\u003e\u003cem\u003ecolleague and friend, NMCP Director Dr.\u0026nbsp;\u003c/em\u003e\u003cem\u003eSamuel Juana Smith\u003c/em\u003e\u003cem\u003e, who passed away unexpectedly while this paper was in preparation. His contributions to fighting malaria in Sierra Leone, and thus to the successful implementation of the work contained herein, were immeasurable.\u003c/em\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eWHO: World malaria report 2022. Geneva: World Health Organization; 2022.\u003c/li\u003e\n\u003cli\u003eWHO: World malaria report 2021. 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I, Agossa FR, Jones CM, Poupardin R, Cornelie S, Akogb\u0026eacute;to M, Ranson H, Corbel V: Molecular characterization of DDT resistance in \u003cem\u003eAnopheles gambiae\u003c/em\u003e from Benin. \u003cem\u003eParasites \u0026amp; vectors \u003c/em\u003e2014, 7:1-9.\u003c/li\u003e\n\u003cli\u003eEdi AVC, N\u0026apos;Dri BP, Chouaibou M, Kouadio FB, Pignatelli P, Raso G, Weetman D, Bonfoh B: First detection of N1575Y mutation in pyrethroid resistant \u003cem\u003eAnopheles gambiae\u003c/em\u003e in Southern C\u0026ocirc;te d\u0026rsquo;Ivoire. \u003cem\u003eWellcome open research \u003c/em\u003e2017, 2.\u003c/li\u003e\n\u003cli\u003eTene B, Poupardin R, Costantini C, Awono-Ambene P, Wondji CS, Ranson H, Antonio-Nkondjio C: Resistance to DDT in an urban setting: common mechanisms implicated in both M and S forms of \u003cem\u003eAnopheles gambiae\u003c/em\u003e in the city of Yaound\u0026eacute; Cameroon. \u003cem\u003ePloS one \u003c/em\u003e2013, 8:e61408.\u003c/li\u003e\n\u003cli\u003eNMCP: Sierra Leone Malaria Indicator Survey. 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World Health Organization; 2017.\u003c/li\u003e\n\u003cli\u003eEssandoh J, Yawson AE, Weetman D: Acetylcholinesterase (Ace-1) target site mutation 119S is strongly diagnostic of carbamate and organophosphate resistance in \u003cem\u003eAnopheles gambiae\u003c/em\u003e ss and \u003cem\u003eAnopheles coluzzii\u003c/em\u003e across southern Ghana. \u003cem\u003eMalaria journal \u003c/em\u003e2013, 12:1-10.\u003c/li\u003e\n\u003cli\u003eDabir\u0026eacute; K, Diabat\u0026eacute; A, Namontougou M, Djogbenou L, Kengne P, Simard F, Bass C, Baldet T: Distribution of insensitive acetylcholinesterase (ace‐1R) in \u003cem\u003eAnopheles gambiae\u003c/em\u003e sl populations from Burkina Faso (West Africa). \u003cem\u003eTropical Medicine \u0026amp; International Health \u003c/em\u003e2009, 14:396-403.\u003c/li\u003e\n\u003cli\u003eElanga-Ndille E, Nouage L, Ndo C, Binyang A, Assatse T, Nguiffo-Nguete D, Djonabaye D, Irving H, Tene-Fossog B, Wondji CS: The G119S acetylcholinesterase (Ace-1) target site mutation confers carbamate resistance in the major malaria vector \u003cem\u003eAnopheles gambiae\u003c/em\u003e from Cameroon: a challenge for the coming IRS implementation. \u003cem\u003eGenes \u003c/em\u003e2019, 10:790.\u003c/li\u003e\n\u003cli\u003eDjogb\u0026eacute;nou L, Weill M, Hougard J-M, Raymond M, Akogbeto M, Chandre F: Characterization of insensitive acetylcholinesterase (ace-1 R) in \u003cem\u003eAnopheles gambiae\u003c/em\u003e (Diptera: Culicidae): resistance levels and dominance. \u003cem\u003eJournal of medical entomology \u003c/em\u003e2007, 44:805-810.\u003c/li\u003e\n\u003cli\u003eAhoua Alou LP, Koffi AA, Adja MA, Tia E, Kouassi PK, Kon\u0026eacute; M, Chandre F: Distribution of ace-1 R and resistance to carbamates and organophosphates in \u003cem\u003eAnopheles gambiae\u003c/em\u003e ss populations from C\u0026ocirc;te d\u0026apos;Ivoire. \u003cem\u003eMalaria Journal \u003c/em\u003e2010, 9:1-7.\u003c/li\u003e\n\u003cli\u003eRaymond M, Berticat C, Weill M, Pasteur N, Chevillon C: Insecticide resistance in the mosquito \u003cem\u003eCulex pipiens\u003c/em\u003e: what have we learned about adaptation? \u003cem\u003eGenetica \u003c/em\u003e2001, 112:287-296.\u003c/li\u003e\n\u003cli\u003eLabb\u0026eacute; P, Berthomieu A, Berticat C, Alout H, Raymond M, Lenormand T, Weill M: Independent duplications of the acetylcholinesterase gene conferring insecticide resistance in the mosquito \u003cem\u003eCulex pipiens\u003c/em\u003e. \u003cem\u003eMolecular Biology and Evolution \u003c/em\u003e2007, 24:1056-1067.\u003c/li\u003e\n\u003cli\u003eAssogba BS, Djogb\u0026eacute;nou LS, Milesi P, Berthomieu A, Perez J, Ayala D, Chandre F, Makoutod\u0026eacute; M, Labb\u0026eacute; P, Weill M: An ace-1 gene duplication resorbs the fitness cost associated with resistance in \u003cem\u003eAnopheles gambiae\u003c/em\u003e, the main malaria mosquito. \u003cem\u003eScientific Reports \u003c/em\u003e2015, 5:14529.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"malaria-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"malj","sideBox":"Learn more about [Malaria Journal](http://malariajournal.biomedcentral.com/)","snPcode":"12936","submissionUrl":"https://submission.nature.com/new-submission/12936/3","title":"Malaria Journal","twitterHandle":"@malariajournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Insecticide resistance, Anopheles gambiae s.l., kdr, Ace-1, Sierra Leone","lastPublishedDoi":"10.21203/rs.3.rs-4720552/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4720552/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eInformation on the status of insecticide resistance in malaria vectors is critical for implementing effective malaria vector control. The Sierra Leone National Malaria Control Program, in collaboration with the PMI VectorLink project, assessed the resistance status to insecticides commonly used in public health, and associated resistance mechanisms in \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l., the main vector of malaria in Sierra Leone.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe susceptibility of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. against pyrethroids with and without piperonyl butoxide (PBO), chlorfenapyr, clothianidin, bendiocarb and pirimiphos-methyl was evaluated in four districts of Sierra Leone in 2018 and 2019 using WHO and CDC bottle bioassay protocols. A subset of samples that were exposed to the insecticides were screened for molecular markers of insecticide resistance, \u003cem\u003eknock-down resistance (kdr)\u003c/em\u003e L1014F, 1014S and N1575Y, and (\u003cem\u003eAce-1\u003c/em\u003e-G119S).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e \u003cem\u003eAnopheles gambiae\u003c/em\u003e s.l. from all sites were resistant to the diagnostic doses of three pyrethroids: deltamethrin, permethrin and alpha-cypermethrin. Intensity of resistance to all three pyrethroids was high, with less than 95% mortality at 10X concentration. However, pre-exposure of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. to PBO increased overall mortality by 41.6%, 50.0% and 44.0% for deltamethrin, permethrin and alpha-cypermethrin, respectively. The vector was susceptible to chlorfenapyr, clothianidin and pirimiphos-methyl, while bendiocarb showed possible resistance. The frequency of \u003cem\u003ekdr\u003c/em\u003e alleles was 98.2% for L1014F, 2.1% for 1014S and 8.9% for N1575Y, while the frequency of the \u003cem\u003eAce-1\u003c/em\u003e G119S allele was 13.6%. Significant deviation from the Hardy-Weinberg equilibrium and deficiency of heterozygotes was detected only at the G119S locus of \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Of the 191 \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. that were molecularly identified to the species level, 81.7% were \u003cem\u003eAn. gambiae\u003c/em\u003e (95% CI 75.3\u0026ndash;86.7), followed by \u003cem\u003eAn. coluzzii\u003c/em\u003e (17.8%, 95% CI (12.8\u0026ndash;24.1) with one hybrid of \u003cem\u003eAn. gambiae/An. coluzzii\u003c/em\u003e 0.5%, 95% CI (0.03\u0026ndash;3.3).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eMalaria vectors were highly resistant to pyrethroids but exposure to PBO partially restored susceptibility in \u003cem\u003eAn. gambiae\u003c/em\u003e s.l. in Sierra Leone. Malaria vectors were susceptible to chlorfenapyr, clothianidin and pirimiphos-methyl with possible resistance to bendiocarb. These data informed the selection and distribution of ITN PBO in Sierra Leone\u0026rsquo;s mass campaigns in 2020 and selection of clothianidin for indoor residual spraying in 2021.\u003c/p\u003e","manuscriptTitle":"Profile and Characterization of Insecticide Resistance Mechanisms in the Anopheles gambiae s.l. 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