First detection of N1575Y mutation in pyrethroid resistant Anopheles gambiae in Southern Cote d'Ivoire.
ABSTRACT: Background. The intensification of insecticide use for both public health and agriculture in Africa has contributed to growing insecticide resistance. Today, resistance to World Health Organization (WHO)-approved insecticide classes is widespread. In an agricultural area of Southern Côte d'Ivoire, the main malaria vector Anopheles coluzzii shows multiple resistance across insecticides mediated by both target site mutation and metabolic mechanisms. To plan new vector control strategies and avert future resistance liabilities caused by cross-resistance mechanisms extant within populations, it is crucial to monitor the development and spread of both resistance and mechanisms. Methods. Larvae of Anopheles gambiae were collected from natural breeding sites in Tiassalé and Elibou, between April and November 2016 and raised to adults . Adult female non-blood fed mosquitoes, three to five days old, were exposed to deltamethrin in WHO bioassays. Extracted DNA samples from exposed mosquitoes were used for species characterisation and genotyping. Results. Most adult An. gambiae tested were resistant to deltamethrin, with mortality rates of only 25% in Tiassalé and 4.4% in Elibou. Molecular analysis of DNA from samples tested showed the presence of both An. coluzzii and An. gambiae s.s in Elibou and only An. coluzzii for Tiassalé. As previously, the L1014F kdr mutation was present at high frequency (79%) in Tiassalé and the L1014S mutation was absent. The N1575Y mutation, which amplifies resistance conferred by L1014F was detected in a single unique individual from a Tiassalé An. coluzzii female whereas in Elibou 1575Y was present in 10 An. gambiae s.s, but not in An. coluzzii. Conclusion. This is the first report of the N1575Y mutation in Côte d'Ivoire, and as in other populations, it is found in both dominant West African malaria vector species. Continued monitoring of N1575Y is underway, as are studies to elucidate its contribution to the resistance of local vector populations.
Project description:Background: To optimize the success of insecticide-based malaria control intervention, knowledge of the distribution of Anopheles gambiae species and insecticide resistance mechanisms is necessary. This paper reported an updated data on pyrethroids/DDT resistance in the An. gambiae s.l population from Togo. Methods: From December 2013 to April 2015, females of indoor-resting An. gambiae s.l were captured in three locations belonging to three different ecological zones. Resistance to DDT, permethrin and deltamethrin was screened in F1 progeny of collected mosquitoes using WHO susceptibility tests. The identification of species of An. gambiae complex and the detection of kdr and ace.1 R allele were carried out using DNA-based molecular techniques. Results:An. gambiae from Kovié and Nangbéto were highly resistant to DDT and permethrin with mortalities rate ranging from 0.83% to 1.58% for DDT and zero to 8.54% for permethrin. Mosquitoes collected in Nangbéto displayed 81.53% mortality with deltamethrin. An. coluzzii and An. gambiae s.s were found in sympatry in Nangbéto and Mango . The allelic frequency of L1014F was high, ranging from 66 to 100% in both An. coluzzii and An. gambiae s.s. For the first time we detected the L1014S allele in both An. coluzzii and An. gambiaes.s. from Togo at the frequency ranging from 5% to 13% in all the sites. The kdr N1575Y was present at various frequencies in both species ranging from 10% to 45%. Both An. gambiae s.s. and An. coluzzii shared the ace1 R mutation in all investigated sites with allelic frequency ranging from 4% to 16%. Conclusion: These results showed that multiple mutations are involved in insecticides resistance in An. gambiae populations from Togo including the kdr L1014F, L1014S, and N1575Y and ace.1 R G119S mutations.
Project description:A key factor affecting malaria vector control efforts in Cameroon is the rapid expansion of insecticide resistance in Anopheles gambiae s.l (An. gambiae) populations; however, mechanisms involved in insecticide resistance in forest mosquito populations are still not well documented yet. The present study was conducted to screen molecular mechanisms conferring insecticide resistance in An. gambiae s.l. populations from the South Cameroon forest region. WHO bioassays were conducted with F0 An. gambiae females aged three to four days from forest (Sangmelima, Nyabessan, and Mbandjock) and urban sites (Yaoundé (Bastos and Nkolondom)), against pyrethroids (permethrin 0.75% and deltamethrin 0.05%) and carbamates (bendiocarb 0.1%). Members of the An. Gambiae s.l. species complex were identified using molecular diagnostic tools. TaqMan assays were used to screen for target site mutations. The expression profiles of eight genes implicated in insecticide resistance were assessed using RT-qPCR. Cuticle hydrocarbon lipids were measured to assess their potential implication in insecticide resistance. Both An. Gambiae and An. coluzzii were detected. An. gambiae was highly prevalent in Sangmelima, Nyabessan, Mbandjock, and Nkolondom. An. coluzzii was the only species found in the Yaoundé city center (Bastos). Low mortality rate to both pyrethroids and bendiocarb was recorded in all sites. High frequency of L1014F allele (75.32-95.82%) and low frequencies of L1014S (1.71-23.05%) and N1575Y (5.28-12.87%) were recorded. The G119S mutation (14.22-35.5%) was detected for the first time in An. gambiae populations from Cameroon. This mutation was rather absent from An. coluzzii populations. The detoxification genes Cyp6m2, Cyp9k1, Cyp6p4, Cyp6z1, as well as Cyp4g16 which catalyzes epicuticular hydrocarbon biosynthesis, were found to be overexpressed in at least one population. The total cuticular hydrocarvbon content, a proxy of cuticular resistance, did not show a pattern associated with pyrethroid resistance in these populations. The rapid emergence of multiple resistance mechanisms in An. Gambiae s.l. population from the South Cameroon forest region is of big concern and could deeply affect the sustainability of insecticide-based interventions strategies in this region.
Project description:BACKGROUND:Mutations in the voltage-gated sodium channel at codon 1014 confer knock-down resistance (kdr) to pyrethroids in a wide range of insects. Anopheles gambiae exhibits two mutant alleles at codon 1014, serine and phenylalanine; and both are now widespread across Africa. Existing screening methods only allow for one resistant allele to be detected per assay. A new locked nucleic acid (LNA) qPCR assay was developed for the simultaneous detection of both mutant alleles and the wild type allele in a single assay. This tri-allelic detection assay was assessed as part of a study of the insecticide resistance in An. gambiae sensu stricto (s.s.) in the previously un-sampled area of Nord Ubangi, Democratic Republic of the Congo. METHODS:Samples from three sites were tested for insecticide susceptibility using WHO bioassays, with and without the synergist PBO preceding pyrethroid exposures, and were subsequently analysed for frequency and resistance-association of the Vgsc-1014 and Vgsc-N1575Y mutations. Results from the LNA-kdr 1014 assay were compared to results from standard TaqMan-kdr assays. RESULTS:Anopheles gambiae sensu lato (s.l.) was by far the predominant vector captured (84%), with only low frequencies of Anopheles funestus s.l. (9%) detected in Nord Ubangi. Molecular identification found An. gambiae s.s. to be the principal vector (99%) although Anopheles coluzzii was detected at very low frequency. Anopheles gambiae were susceptible to the carbamate insecticide bendiocarb, but resistant to DDT and to the pyrethroids permethrin and deltamethrin. Susceptibility to both pyrethroids was partially restored with prior exposure to PBO suggesting likely involvement of metabolic resistance. Anopheles gambiae s.s. was homozygous for kdr resistant alleles with both the L1014F and L1014S mutations present, and the N1575Y polymorphism was present at low frequency. The LNA-kdr assay simultaneously detected both resistant alleles and gave results entirely consistent with those from the two TaqMan-kdr assays. CONCLUSION:This study provides rare data on insecticide resistance and mechanisms in Anopheles from the centre of Africa, with the first detection of N1575Y. Nord Ubangi populations of An. gambiae s.s. show insecticide resistance mediated by both metabolic mechanisms and Vgsc mutations. The LNA-kdr assay is particularly suitable for use in populations in which both 1014S and 1014F kdr alleles co-occur and provides robust results, with higher throughput and at a quarter of the cost of TaqMan assays.
Project description:The emergence and spread of insecticide resistance in the major African malaria vectors Anopheles gambiae (s.s.) and An. arabiensis may compromise the current vector control interventions and threatens the global malaria control and elimination efforts.Insecticide resistance was monitored in several study sites in Ethiopia from 2013 to 2015 using papers impregnated with discriminating concentrations of DDT, deltamethrin, bendiocarb, propoxur, malathion, fenitrothion and pirimiphos-methyl, following the WHO insecticide susceptibility test procedure. Mosquitoes sampled from different localities for WHO bioassay were morphologically identified as An. gambiae (s.l.) using standard taxonomic keys. Samples were identified to species using species-specific polymerase chain reaction (PCR) and screened for the presence of target site mutations L1014F, L1014S and N1575Y in the voltage gated sodium channel (VGSC) gene and G119S in the acethylcholinesterase (AChE) gene using allele-specific PCR. Biochemical assays were performed to assess elevated levels of acetylcholinesterases, carboxylcholinesterases, glutathione-S-transferases (GSTs) and cytochrome P450s monooxygenases in wild populations of An. arabiensis, compared to the fully susceptible Sekoru An. arabiensis laboratory strain.Populations of An. arabiensis were resistant to DDT and deltamethrin but were susceptible to fenitrothion in all the study sites. Reduced susceptibility to malathion, pirimiphos-methyl, propoxur and bendiocarb was observed in some of the study sites. Knockdown resistance (kdr L1014F) was detected in all mosquito populations with allele frequency ranging from 42 to 91%. Elevated levels of glutathione-S-transferases (GSTs) were detected in some of the mosquito populations. However, no elevated levels of monooxygenases and esterases were detected in any of the populations assessed.Anopheles arabiensis populations from all surveyed sites in Ethiopia exhibited resistance against DDT and pyrethroids. Moreover, some mosquito populations exhibited resistance to propoxur and possible resistance to bendiocarb. Target site mutation kdr L1014F was detected in all mosquito populations while elevated levels of glutathione-S-transferases (GSTs) was detected in some mosquito populations. The reduced susceptibility of An. arabiensis to propoxur and bendiocarb, which are currently used for indoor residual spraying (IRS) in Ethiopia, calls for continuous resistance monitoring, in order to plan and implement evidence based insecticide resistance management.
Project description:Members of the Anopheles gambiae (s.l.) complex are one of the major vectors of malaria in Africa. LLINs and IRS are the most effective tools used in vector control of malaria. However, their effectiveness may be hampered by the development and spread of insecticide resistance in the target vectors species. The objective of this study was to assess the susceptibility of Anopheles gambiae (s.l.) mosquitoes from South-West Cameroon to deltamethrin, permethrin and to malathion, four years after the mass deployment of LLINs.Anopheles larvae were collected from Limbe, Tiko and Buea, three cities of the Fako division and reared until adult emergence. Adult mosquitoes from field larvae were identified as belonging to the Anopheles gambiae (s.l.) complex using standard identification keys. Susceptibility of mosquito samples to deltamethrin, permethrin and malathion was assessed using WHO susceptibility tests protocol for adult mosquitoes. Molecular identification of tested samples was performed using the PCR SINE200 protocol and by PCR-RFLP. The kdr alleles were genotyped using the hot ligation oligonucleotide assay (HOLA).Two species of the An. gambiae (s.l.) complex, An. coluzzii and An. gambiae (s.s.) were identified in all three study locations with high proportions of An. coluzzii in Limbe (84.06%) and Tiko (92.2%), while in Buea, An. coluzzii (55.6%) and An. gambiae (s.s.) (44.4%) occurred almost in the same proportions. Tested samples were found resistant to pyrethroids (deltamethrin and permethrin) in all locations (< 90% mortality), with > 3-fold increase of KDT50 values compared with the Kisumu susceptible reference strain of An. gambiae (s.s.). However, the mosquito populations from Limbe and Buea were fully susceptible to malathion. The L1014F kdr was found in both An. coluzzii and An. gambiae (s.s.) with the highest frequencies found in An. gambiae (s.l.) populations from Tiko (94%) and Buea (90%) compared with the Limbe population (66%) (P = 0.00063, df = 2). No kdr L1014S was observed in analyzed samples.These findings reemphasize the ongoing development of An. gambiae (s.l.) resistance to pyrethroids used in impregnating LLINs and suggest the use of malathion as an alternative insecticide for IRS in complementarity with LLINs.
Project description:BACKGROUND:In recent years, the scale-up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has greatly reduced malaria transmission. However, malaria remains a global public health concern with the majority of the disease burden in sub-Saharan Africa. Insecticide resistance is a growing problem among Anopheles vector populations, with potential implications for the continued effectiveness of available control interventions. Improved understanding of current resistance levels and underlying mechanisms is essential to design appropriate management strategies and to mitigate future selection for resistance. METHODS:Anopheles gambiae sensu lato mosquitoes were collected from three villages in Faranah Prefecture, Guinea and their levels of susceptibility to seven insecticides were measured using CDC resistance intensity bioassays. Synergist assays with piperonyl butoxide (PBO) were also undertaken to assess the role of elevated mixed-function oxidases in resistance. Five hundred and sixty-three mosquitoes underwent molecular characterization of vector species, presence of target site mutations (L1014F kdr, N1575Y and G119S Ace-1), Plasmodium falciparum infection, and relative expression of three metabolic genes (CYP6M2, CYP6P3 and GSTD3). RESULTS:In Faranah, resistance to permethrin and deltamethrin was observed, as well as possible resistance to bendiocarb. All assayed vector populations were fully susceptible to alpha-cypermethrin, pirimiphos-methyl, clothianidin and chlorfenapyr. Plasmodium falciparum infection was detected in 7.3% (37/508) of mosquitoes tested. The L1014F kdr mutation was found in 100% of a sub-sample of 60 mosquitoes, supporting its fixation in the region. The N1575Y mutation was identified in 20% (113/561) of individuals, with ongoing selection evidenced by significant deviations from Hardy-Weinberg equilibrium. The G119S Ace-1 mutation was detected in 62.1% (18/29) of mosquitoes tested and was highly predictive of bendiocarb bioassay survival. The metabolic resistance genes, CYP6M2, CYP6P3 and GSTD3, were found to be overexpressed in wild resistant and susceptible An. gambiae sensu stricto populations, compared to a susceptible G3 colony. Furthermore, CYP6P3 was significantly overexpressed in bendiocarb survivors, implicating its potential role in carbamate resistance in Faranah. CONCLUSIONS:Identification of intense resistance to permethrin and deltamethrin in Faranah, is of concern, as the Guinea National Malaria Control Programme (NMCP) relies exclusively on the distribution of pyrethroid-treated LLINs for vector control. Study findings will be used to guide current and future control strategies in the region.
Project description:BACKGROUND:Insecticide resistance in Anopheles coluzzii mosquitoes has become widespread throughout West Africa including in Burkina Faso. The insecticide resistance allele (kdr or L1014F) is a prime indicator that is highly correlated with phenotypic resistance in West Africa. Studies from Benin, Ghana and Mali have suggested that the source of the L1014F is introgression of the 2L divergence island via interspecific hybridization with Anopheles gambiae. The goal of this study was to characterize local mosquito populations in the Nouna Department, Burkina Faso with respect to: (i) the extent of introgression between An. coluzzii and An. gambiae, (ii) the frequency of the L1014F mutation and (iii) Plasmodium infection rates. METHODS:A total of 95 mosquitoes were collected from ten sites surrounding Nouna town in Kossi Province, Burkina Faso in 2012. The species composition, the extent of introgression in An. coluzzii mosquitoes and their Plasmodium infection rates were identified with a modified version of the "Divergence Island SNP" (DIS) genotyping assay. RESULTS:The mosquito collection contained 70.5% An. coluzzii, 89.3% of which carried a 3 Mb genomic region on the 2L chromosome with L1014F insecticide resistance mutation that was introgressed from An. gambiae. In addition, 22.4% in the introgressed An. coluzzii specimens were infected with Plasmodium falciparum, whereas none of the non-introgressed ("pure") An. coluzzii were infected. CONCLUSION:This paper is the first report providing divergence island SNP genotypes for natural population of Burkina Faso and corresponding Plasmodium infection rates. These observations warrant further study and could have a major impact on future malaria control strategies in Burkina Faso.
Project description:Bioassays and molecular diagnostics are routinely used for the monitoring of malaria vector populations to support insecticide resistance management (IRM), guiding operational decisions on which insecticides ought to be used for effective vector control. Previously developed TaqMan assays were optimised to distinguish the wild-type L1014 from the knockdown resistance (kdr) point mutations 1014F and 1014S (triplex reaction), and the N1575 wild-type from the point mutation 1575Y (duplex reaction). Subsequently, artificial pools of Anopheles gambiae (An. gambiae) specimens with known genotypes of L1014F, L1014S, and N1575Y were created, nucleic acids were extracted, and kdr mutations were detected. These data were then used to define a linear regression model that predicts the allelic frequency within a pool of mosquitoes as a function of the measured ?Ct values (Ct mutant - Ct wild type probe). Polynomial regression models showed r2 values of >0.99 (p < 0.05). The method was validated with populations of variable allelic frequencies, and found to be precise (1.66?2.99%), accurate (3.3?5.9%), and able to detect a single heterozygous mosquito mixed with 9 wild type individuals in a pool of 10. Its pilot application in field-caught samples showed minimal differences from individual genotyping (0.36?4.0%). It allowed the first detection of the super-kdr mutation N1575Y in An. gambiae from Mali. Using pools instead of individuals allows for more efficient resistance allele screening, facilitating IRM.
Project description:BACKGROUND:Vector control activities, namely long-lasting insecticidal nets (LLIN) and indoor residual spraying (IRS), have contributed significantly to the decreasing malaria burden observed in The Gambia since 2008. Nevertheless, insecticide resistance may threaten such success; it is important to regularly assess the susceptibility of local malaria vectors to available insecticides. METHODS:In the transmission seasons of 2016 and 2017, Anopheles gambiae (s.l.) larvae were sampled in or around the nine vector surveillance sentinel sites of the Gambia National Malaria Control Programme (GNMCP) and in a few additional sampling points. Using WHO susceptibility bioassays, female adult mosquitoes were exposed to insecticide-impregnated papers. Molecular identification of sibling species and insecticide resistance molecular markers was done on a subset of 2000 female mosquitoes. RESULTS:A total of 4666 wild-caught female adult mosquitoes were exposed to either permethrin (n = 665), deltamethrin (n = 744), DDT (n = 1021), bendiocarb (n = 990) or pirimiphos-methyl (n = 630) insecticide-impregnated papers and control papers (n = 616). Among the 2000 anophelines, 1511 (80.7%) were Anopheles arabiensis, 204 (10.9%) Anopheles coluzzii, 75 (4%) Anopheles gambiae (s.s.), and 83 (4.4%) An. gambiae (s.s.) and An. coluzzii hybrids. There was a significant variation in the composition and species distribution by regions and year, P = 0.009. Deltamethrin, permethrin and DDT resistance was found in An. arabiensis, especially in the coastal region, and was mediated by Vgsc-1014F/S mutations (odds ratio = 34, P = 0.014). There was suspected resistance to pirimiphos-methyl (actellic 300CS) in the North Bank Region although only one survivor had the Ace-1-119S mutation. CONCLUSIONS:As no confirmed resistance to bendiocarb and actellic 300CS was detected, the national malaria control programme can continue using these insecticides for IRS. Nevertheless, the detection of Ace-1 119S mutation warrants extensive monitoring. The source of insecticide pressure driving insecticide resistance to pyrethroids and DDT detected at the coastal region should be further investigated in order to properly manage the spread of resistance in The Gambia.
Project description:Insecticide resistance across sub-Saharan Africa may impact the continued effectiveness of malaria vector control. We investigated the association between carbamate and pyrethroid resistance with Anopheles gambiae s.l. parity, Plasmodium falciparum infection, and molecular insecticide resistance mechanisms in Guinea. Pyrethroid resistance was intense, with field populations surviving ten times the insecticidal concentration required to kill susceptible individuals. The L1014F kdr-N1575Y haplotype and I1527T mutation were significantly associated with mosquito survival following permethrin exposure (Prevalence Ratio; PR?=?1.92, CI?=?1.09-3.37 and PR?=?2.80, CI?=?1.03-7.64, respectively). Partial restoration of pyrethroid susceptibility following synergist pre-exposure suggests a role for mixed-function oxidases. Carbamate resistance was lower and significantly associated with the G119S Ace-1 mutation. Oocyst rates were 6.8% and 4.2% among resistant and susceptible mosquitoes, respectively; survivors of bendiocarb exposure were significantly more likely to be infected. Pyrethroid resistant mosquitoes had significantly lower parity rates than their susceptible counterparts (PR?=?1.15, CI?=?1.10-1.21). Our findings emphasize the need for additional studies directly assessing the influence of insecticide resistance on mosquito fitness.