Organophosphate and carbamate susceptibility profiling of Anopheles gambiae sl. across different ecosystems in southern Benin

Background To overcome the spread of high pyrethroid resistance in the main malaria vectors and malaria disease persistence, it is crucial to look for effective and better resistance management strategies. Understanding the phenotypic profile of Anopheles gambiae sl. against alternatives insecticides like organophosphates and carbamates is crucial. Methods Anopheles larvae and pupae were collected from the breeding sites in rice fields, pineapple crop areas, and peri-urban areas. WHO susceptibility tests were conducted on unfed female mosquitoes aged 3–5 days old. Mosquitoes were exposed to malathion 5%, pirimiphos-methyl 0.25%, and bendiocarb 0.1% using the standard WHO protocol. Polymerase chain reaction (PCR) techniques were used to detect species, kdr and Ace-1 mutations. Results Anopheles gambiae sl. from Sèdjè-Dénou rice field population was resistant to bendiocarb (0.1%) with a mortality rate of 72.2% whereas Anopheles gambiae sl. populations from Zinvié-Dokomey (rice field), Zè-Tozounmè (pineapple field), and Adjagbo (peri-urban area) were suspected to be resistant with mortality rates of 90%, 93.5%, 95.4% respectively. However, all of them were susceptible to organophosphates (malathion and pirimiphos-methyl) with a mortality rate of 100%. PCR assay revealed that 100% of the mosquitoes tested were Anopheles coluzzii. The frequencies of Ace-1R mutation in all Anopheles coluzzii populations tested were low (3–27%). Conclusions Organophosphates (malathion and pirimiphos-methyl) have maintained their efficacy against Anopheles coluzzii populations from Sèdjè-Dénou (rice field), Zè Tozounmè (pineapple field), Zinvié Dokomey (rice field), or Adjagbo (peri-urban area). The good efficacy of these organophosphates against Anopheles coluzzii populations from the southern part of Benin are observed in the current study. The use of pirimiphos-methyl for IRS in this part of the country would be a successful alternative for malaria control in this area.

BackgroundThe detection of insecticide resistance in natural populations of Anopheles vectors is absolutely necessary for malaria control. In the African region, the WHO insecticide susceptibility test is the most common method for assessing resistance status. In order to search for a simple, rapid and more reliable technique in the assessment of insecticide resistance in malaria vectors, we compared the WHO tests with the CDC bottle bioassay in the Ouemé province of southern Benin where insecticide resistance has been widely reported.MethodsLarvae and pupae of Anopheles gambiae s.l. mosquitoes were collected from the breeding sites in Ouemé. WHO and CDC susceptibility tests were conducted simultaneously on unfed female mosquitoes aged 2–5 days old. WHO bioassays were performed with impregnated papers of deltamethrin (0.05%) and bendiocarb (0.1%), whereas CDC bioassays were performed with stock solutions of deltamethrin (12.5 μg per bottle) and bendiocarb (12.5 μg per bottle). PCR techniques were used to detect species, Kdr and Ace-1 mutations. CDC biochemical assays using synergists were also conducted to assess the metabolic resistance.ResultsA slight decrease in mortality rates was observed with 97.95% and 98.33% obtained from CDC and WHO bioassays respectively in populations of mosquitoes from Adjara and Dangbo. PCR revealed that all specimens tested were Anopheles gambiae s.s. The Kdr mutation was found at high frequency in all populations and both the Kdr mutation and mono-oxygenase enzymes were implicated as mechanisms of pyrethroid resistance in An. gambiae from Misserete.ConclusionThis study emphasizes that both WHO and CDC bioassays give similar results with regards to the susceptibility of mosquitoes to insecticides in southern Benin. There were complementarities between both methods, however, some specificity was noted for each of the two methods used. Both Kdr and metabolic mechanisms were implicated in the resistance.

BackgroundNeonicotinoids are gradually being introduced into malaria vector control due to their mode of action that targets insect nicotinic acetylcholine receptors (nAChRs). However, prior to scaling up novel insecticides, it is important to assess the susceptibility of the mosquito population to this molecule. The present study aimed to determine the susceptibility level of mosquitos collected from agricultural and urban sites against neonicotinoids in Benin.Material and methodsAn. gambiae sl. larvae were collected from three urban sites and two agricultural sites in southern Benin. An. gambiae sl. larvae collected from three urban and two agricultural sites in southern Benin were exposed to neonicotinoids (clothianidin, acetamiprid, and imidacloprid) for 1 hour, and mortality rates were monitored from 24h to day 7 post-exposure. After molecular identification of the samples, Taqman assays were used to genotype key pyrethroid-resistant markers, and qPCRs were performed to establish the level of expression of detoxification enzymes in resistant mosquitoes.ResultsOverall, resistance to acetamiprid and imidacloprid was observed in the five study sites surveyed whereas full susceptibility was recorded to clothianidin. Anopheles coluzzii was the only species found in all sites. TaqMan genotyping showed a very low presence of key pyrethroid-resistant markers suggesting that metabolic resistance is more implicated in neonicotinoid resistance in Anopheles. coluzzii populations from southern Benin. qPCR performed revealed overexpression of P450 genes (CYP6M2, CYP6P4, CYP6Z1, CYP6Z2, and CYP4G16) and sensory proteins (Sap1, Sap2, and Sap3).ConclusionThis first assessment of neonicotinoid resistance in Benin showed evidence of neonicotinoid resistance in both agricultural and urban areas. However, full susceptibility to clothianidin was observed, showing that this insecticide could be used to enhance the efficacy of control tools. In addition, there is a need of an integrated approach, including coordinated education on pesticide use, to improve the overall management of insecticide resistance in both agricultural and public health sectors.

Anopheles gambiae, which is the main malaria vector in Benin has developed high level of resistance to pyrethroid insecticides. This raises serious concerns to the future use of long- lasting insecticidal nets (LLIN) and indoor residual spraying (IRS). It is therefore important to seek better and effective resistance management strategies which will use organophosphates or carbamates as alternatives against pyrethroid resistant malaria vectors in the field. Larvae and pupae of A. gambiae s.l. mosquitoes were collected from the breeding sites in Dassa-Zoume and Zogbodomey districts. WHO susceptibility tests were conducted on unfed female mosquitoes aged 2-5 days old. WHO bioassays were performed with impregnated papers with fenitrothion 1%, pirimiphos-methyl 0.25%, and bendiocarb 0.1%. Polymerase chain reaction (PCR) techniques were used to detect species and Ace-1 mutations. A. gambiae Dassa-Zoume populations were susceptible to bendiocarb 0.1% with mortality rate of 99%. A. gambiae Zogbodomey populations were susceptible to pirimiphos-methyl 0.25% and fenitrothion 1% with mortality rates of 98.96 and 99%, respectively. PCR assay revealed that 100% of mosquitoes tested were A. gambiae s.s. The frequencies of Ace-1R mutation in A. gambiae Dassa-Zoume and Zogbodomey were 0%. Carbamates (bendiocarb) and organophosphates (fenitrothion and pirimiphos-methyl) have maintained their efficiency against A. gambiae Dassa-Zoume and Zogbodomey populations. Carbamates (bendiocarb) and organophosphates (fenitrothion and pirimiphos-methyl) have proven to be powerful alternatives against pyrethroid resistant malaria vectors such as A. gambiae Dassa-Zoume and Zogbodomey populations. The use of any of these three compounds in the centre Benin would be successful in malaria vector control.   Key   words:   Anopheles gambiae,   Ace-1,   resistance,   fenitrothion,   pirimiphos-methyl, bendiocarb, Benin.

High malaria prevalence remains a major problem in Ruangwa District, despite the high coverage rates of malaria control interventions. The objective of this study was to assess the level of insecticide resistance in malaria vectors in southern, Tanzania. The study was conducted in three villages of Likangara, Nandagara and Chienjere. Resistance level to insecticides in An. gambiae s.l was evaluated using a standard WHO Susceptibility Test Kit. These mosquitoes were reared from larvae collected in varoius breeding sites in a major rice, vegetable and leguminous plant cultivation area in which pesticides use is intensive. Each test was run in four replicates of 25 adult non-blood fed female Anopheles gambiae s.l mosquitoes per tube. In total, 100 specimens were exposed for standard concentration of 1% fenitrothion, 0.1% bendiocarb, 0.75% permethrin and 0.05% deltamethrin using WHO susceptibility test kit. The number of knocked down mosquitoes were recorded at 10,15,20,30,50 and 60 min and mortality rates were determined after 24 hours and results were classified according to WHO guidelines, as susceptible (97-100%), possible resistance (90%-98%) and resistance (<90%). The results indicate suggestive levels of mosquitoes resistance to Bendiocarb at (75%), possible resitance levels of Deltamethrin at (95%), Permethrin at (97%) and Fenitrothion at (96%) (p < 0.05). This implies that there is reduced effectiveness of insecticides used in vector control interventions in the district. Superior insecticides should made available and introduced in order to promote the sustainable management of malaria vectors and elimination malaria transmission in the district.

BackgroundIn Myanmar, three types of artemisinin-based combination therapy (ACT) are recommended as first-line treatment of uncomplicated falciparum malaria: artemether–lumefantrine (AL), artesunate–mefloquine (AS + MQ), and dihydroartemisinin–piperaquine (DP). Resistance to both artemisinins and ACT partner drugs has been reported from the Greater Mekong Sub-region, and regular efficacy monitoring of the recommended ACT is conducted in Myanmar. This paper reports on results from studies to monitor the efficacy of the three forms of ACT in sentinel sites in northern Myanmar, and investigations of mutations in the Kelch13 (k13) propeller domain.MethodsSeven therapeutic efficacy studies were conducted in 2011–12 and 2014 in three sentinel sites in Myanmar (Tamu, Muse, Tabeikkyin). Three studies were done for the evaluation of AL (204 patients), two studies for AS + MQ (119 patients) and two studies for DP (147 patients). These studies were done according to 2009 standard WHO protocol. Polymorphisms in the k13 propeller domain were examined in dried blood spots collected on day 0. The primary endpoint was adequate clinical and parasitological response (ACPR) on day 28 for AL and on day 42 for DP and AS + MQ, corrected to exclude re-infection using polymerase chain reaction (PCR) genotyping. Safety data were collected through self-reporting.ResultsPCR-corrected ACPR was 97.2–100% for AL, 98.6–100% for AS + MQ and 100% for DP across the study sites and years. All studies found a prevalence of k13 mutations (>440) above 23% in the day-0 samples. The F446I mutation was the most common mutation, making up 66.0% of the mutations found. Seven out of nine day-3 positive patients were infected with k13 wild type parasites. The remaining two cases with day-3 parasitaemia had the P574L mutation.ConclusionsThe efficacy of AL, AS + MQ and DP remains high in northern Myanmar despite widespread evidence of k13 mutations associated with delayed parasite clearance. This study showed that already in 2012 there was a high frequency of k13 mutations in Myanmar on the border with India. The high efficacy of the recommended ACT gives confidence in the continued recommendation of the use of these treatments in Myanmar.Trial registration numbers ACTRN12611001245987 (registered 06-12-2011) and ACTRN12614000216617 (registered 28-02-2014)

Multiple insecticide resistance mechanisms in urban population of Anopheles coluzzii (Diptera: culicidae) from Lagos, South-West Nigeria

Resistance of Plasmodium falciparum to antimalarial drugs in a highly endemic area of southern Viet Nam: a study in vivo and in vitro

BackgroundPyrethroids are the main insecticides used in vector control for malaria. However, their extensive use in the impregnation of long-lasting insecticidal nets (LLINs) and indoor residual spraying has led to the development of resistance, threatening its success as a tool for malaria control. Baseline data prior to large scale distribution of LLINs are important for the implementation of efficient strategies. However, no data on the susceptibility of malaria vectors is available in the Moyen-Ogooué Province in Gabon. The aim of this study was to assess the susceptibility to pyrethroids and organochlorides of malaria vectors from a semi-urban and rural areas of the province and to determine the frequency of insecticide resistance genes.MethodsLarvae were collected from breeding sites in Lambaréné and Zilé and reared to adults. Three to five-day old female Anopheles gambiae sensu lato mosquitoes were used in cone tube assays following the WHO susceptibility tests protocol for adult mosquitoes. A subsample was molecularly identified using the SINE200 protocol and the frequency of Vgsc-1014 F and − 1014 S mutations were determined.ResultsAnopheles gambiae sensu stricto (s.s.) was the sole species present in both Lambaréné and Zilé. Mosquito populations from the two areas were resistant to pyrethroids and organochlorides. Resistance was more pronounced for permethrin and DDT with mortality lower than 7% for both insecticides in the two study areas. Mosquitoes were statistically more resistant (P < 0.0001) to deltamethrin in Lambaréné (51%) compared to Zilé (76%). All the mosquitoes tested were heterozygous or homozygous for the knockdown resistance (Kdr) mutations Vgsc-L1014F and Vgsc-L1014S with a higher proportion of Vgsc-L1014F homozygous in Lambaréné (76.7%) compared to Zilé (57.1%).ConclusionThis study provides evidence of widespread resistance to pyrethroids in An. gambiae s.s., the main malaria vector in the Moyen-Ogooué Province. Further investigation of the mechanisms underlining the resistance of An. gambiae s.s. to pyrethroids is needed to implement appropriate insecticide resistance management strategies.

A total of 180 larval collection sites (e.g., rice paddies, marshes, ground pools, ponds, stream margins, and irrigation and drainage ditches) was surveyed within a 2 km radius from Warrior Base training area, 5 km south of Panmunjeom (Joint Security Area, demilitarized zone), Gyeonggi Province, Republic of Korea (ROK), from May through October, 2007 to characterize larval habitat distributions of members of the Anopheles Hyrcanus Group (An. sinensis, An. lesteri, An. pullus, An. belenrae, An. kleini, and An. sineroides). A total of 5,859 anopheline larvae was collected from 84.4% of the sites surveyed, of which 4,071 were identified to species by polymerase chain reaction (PCR) using the ribosomal DNA internal transcribed spacer 2 (rDNA ITS2). Anopheles sinensis (52.6%) was the most frequently collected, followed by An. kleini (29.4%), An. sineroides (9.8%), An. pullus (6.7%), An. belenrae (1.1%), and An. lesteri (0.5%). Anopheles pullus and An. kleini were collected in greater proportions in May and from May - July, respectively. Few An. sinensis were collected from May - June, but it was the predominant species collected by August, and accounted for >80% of all larvae from September - October. Anopheles kleini was found in all habitats sampled; however, it was collected most frequently in young growth rice paddies, while An. sinensis was collected more frequently in mature and post-harvest paddies. Anopheles pullus was associated with pre-cultivated rice paddies, including water-filled tire ruts left from the previous fall's harvest.

BackgroundIn 2011, Benin’s National Malaria Control Programme (NMCP) organized a nationwide mass distribution campaign of LLINs throughout the country. Following this intervention, it was important to assess whether the level of susceptibility of malaria vectors to insecticides had remained the same as compared to the pre-intervention period. The current study investigated this.MethodsLarval collections were conducted in Ifangni, Sakété, Pobè and Kétou districts located in Plateau department, Southeastern Benin before (2009) and after (2012–2013) LLIN distribution. Anopheles gambiae sensu lato (s.l.) larvae from the 4 study districts were reared to adulthood and WHO susceptibility tests were conducted. The insecticides tested were deltamethrin (0.05%), permethrin (0.75%), bendiocarb (0.1%) and DDT (4%). Molecular species identification as well as, the characterization of the kdr L1014F mutation were also performed in the An. gambiae s.l. complex using PCR method.ResultsOverall, a significant decrease in mortality rates of An. gambiae s.l. to deltamethrin (0.05%), permethrin (0.75%) and DDT (4%) was observed post-LLIN distribution, respectively: (100% vs 80.9%, p < 0.0001), (77.5% vs 70%, p = 0.01) and, (47.8% vs 4.4%, p < 0.0001). By contrast, susceptibility of vectors to bendiocarb (0.1%) remained the same (100% mortality in the WHO susceptibility tube tests) pre- and post-intervention. An increase in the kdr L1014F frequency was observed post-LLIN distribution [F(kdr) = 0.91)] compared to the pre-intervention period [F(kdr) = 0.56], p < 0.0001. Anopheles coluzzii and An. gambiae were the two molecular species identified in the study area.ConclusionThe decrease susceptibility to pyrethroids and DDT as well as, the increase in the frequency of the kdr L1014F mutation after the intervention stressed at the time, the need for the development and implementation of effective insecticide resistance management strategies. At present, an update of the vectors resistance status in the area is also necessary for decision-making.

This study aimed to carry out a malaria situation analysis, species composition and susceptibility levels of the main malaria vector, Anopheles stephensi, to different insecticides in Bashagard. A longitudinal survey was conducted in 2 randomly selected villages in Bashagard. Malaria vectors were sampled by dipping method for the larvae and hand catch, night-biting catch, total catch, and shelter pit collection for the adults. Standard WHO susceptibility tests were used for a variety of insecticides on F1 progeny of An. stephensi reared from wild-caught females. In total, 693 adult anopheline mosquitoes and 839 third and fourth-instar larvae were collected and identified. They comprised 7 species; the most abundant adult and larvae anopheline mosquito was An. dthali (40.7% and 30.5% respectively). An. culicifacies (24.2%) and An. stephensi (16.7%) were the next most common species for adult mosquitoes. An. stephensi was fully susceptible to malathion and pyrethroid insecticides but resistant to DDT and tolerant to dieldrin.

BackgroundKnowing the extent and spread of insecticide resistance in malaria vectors is vital to successfully manage insecticide resistance in Africa. This information in the main malaria vector, Anopheles funestus sensu stricto, is completely lacking in the most populous country in Africa, Nigeria. This study reports the insecticide susceptibility status and the molecular basis of resistance of An. funestus as well as its involvement in malaria transmission in Akaka-Remo, a farm settlement village in southwest Nigeria.ResultsPlasmodium infection analysis using TaqMan protocol coupled with a nested PCR revealed an infection rate of 8% in An. funestus s.s. from Akaka-Remo. WHO susceptibility tests showed this species has developed multiple resistance to insecticides in the study area. Anopheles funestus s.s. population in Akaka-Remo is highly resistant to organochlorines: dieldrin (8%) and DDT (10%). Resistance was also observed against pyrethroids: permethrin (68%) and deltamethrin (87%), and the carbamate bendiocarb (84%). Mortality rate with DDT slightly increased (from 10 to 30%, n = 45) after PBO pre-exposure indicating that cytochrome P450s play little role in DDT resistance while high mortalities were recorded after PBO pre-exposure with permethrin (from 68 to 100%, n = 70) and dieldrin (from 8 to 100%, n = 48) suggesting the implication of P450s in the observed permethrin and dieldrin resistance. High frequencies of resistant allele, 119F in F0 (77%) and F1 (80% in resistant and 72% in susceptible) populations with an odd ratio of 1.56 (P = 0.1859) show that L119F-GSTe2 mutation is almost fixed in the population. Genotyping of the A296S-RDL mutation in both F0 and F1 samples shows an association with dieldrin resistance with an odd ratio of 81 (P < 0.0001) (allelic frequency (R) = 76% for F0; for F1, 90 and 10% were observed in resistant and susceptible populations, respectively) as this mutation is not yet fixed in the population.ConclusionThe study reports multiple insecticide resistance in An. funestus from Akaka Remo. It is, therefore, necessary to pay more attention to this major malaria vector for effective malaria control in Nigeria.

BackgroundMalaria vector control in Tanzania is based on use of long-lasting insecticide treated nets (LLINs) and indoor residual spraying (IRS), which both rely on the use of chemical insecticides. The effectiveness of these control tools is endangered by the development of insecticide resistance in the major malaria vectors. This study was carried out to monitor the susceptibility status of major malaria vectors to insecticides used for IRS and LLINs in mainland Tanzania.MethodsMosquito larvae were collected in 20 sites of Tanzania mainland in 2015. Phenotypic resistance was determined using standard WHO susceptibility tests. Molecular assay were used to determine distribution of Anopheles gambiae sub-species. A microplate assay approach was used for identifying enzyme levels on single mosquitoes from each sites compared with a susceptible reference strain, An. gambiae sensu stricto (s.s.) Kisumu strain.ResultsAnopheles arabiensis was the dominant malaria specie in the country, accounting for 52% of the sibling species identified, while An. gambiae s.s. represented 48%. In Arumeru site, the dominant species was An. arabiensis, which was resistant to both pyrethroids (permethrin and deltamethrin), and pirimiphos-methyl, and had significant elevated levels of GSTs, non-specific esterases, and oxidase enzymes. An. arabiensis was also a dominant species in Kilombero and Kondoa sites, both were resistant to permethrin and deltamethrin with significant activity levels of oxidase enzymes. Resistance to bendiocarb was recorded in Ngara site where specie composition is evenly distributed between An. gambiae s.s. and An.arabiensis. Also bendiocarb resistance was recorded in Mbozi site, where An. gambiae s.s. is the dominant species.ConclusionsOverall, this study confirmed resistance to all four insecticide classes in An. gambiae sensu lato in selected locations in Tanzania. Results are discussed in relation to resistance mechanisms and the optimization of resistance management strategies.

BackgroundThe current study was undertaken to determine the optimal wash-drying regimen and the effects of different washing procedures on the efficacy, and durability of four brands of newly introduced long-lasting insecticide-treated nets (LLINs) under tropical conditions.MethodsIn the current study, the following four LLINs were tested: Olyset®, PermaNet ®2.0, BASF® and TNT®. Nets were divided into three sets; one set was washed by hand rubbing and air-dried either hanging or spread on the ground in direct sunlight or hanging or spread on the ground under the shade. A second set was washed using the WHO protocol (machine) and the third set was washed by beating the nets on rocks. The biological activities of the nets were assessed by a three-minute bioassay cone test and the residual insecticide contents were determined using high performance liquid chromatography (HPLC) procedure.ResultsNets that were dried hanging under the shade retained more insecticide, 62.5% and recorded higher mortality compared to nets which were dried lying on the ground in direct sunlight 58.8%, nets dried under the shade spread on the ground 56.3%, and 57.8% for nets dried hanging in direct sunlight. It was also observed that nets washed by the standard WHO protocol, retained more insecticide and were more effective in killing mosquitoes compared to nets washed by local methods of hand rubbing and beating on rocks. There were significant differences between drying regimens (p < 0.0001) and between washing procedures (p < 0.001) respectively. However, the effect of net type was statistically insignificant. The statistical differences on individual nets were also compared, for PermaNet® and TNT there were no significant differences observed between the four drying regimens (p = 0.7944 and 0.4703) respectively). For BASF and Olyset, the differences were significant (p < 0.001 and p > 0.0001).ConclusionThe results of this study suggest that washing and drying regimen influence the insecticidal activity of LLINs. The standard WHOPES washing protocol underestimates the amount of insecticide washed from LLINs compared to the abrasive washing procedures that are used in the field. This suggests that there is need to educate net users to adopt a more gentle washing procedure while handling LLINs. The education should accompany net distribution campaigns.