Kdr Frequency Research Articles

Primary mammary angiosarcomas harbor frequent mutations in KDR and PIK3CA and show evidence of distinct pathogenesis.

Angiosarcoma (AS) is the most frequent primary sarcoma of the breast but nevertheless remains uncommon, accounting for <0.05% of breast malignancies. Secondary mammary AS arise following radiation therapy for breast cancer, in contrast to primary AS which occur sporadically. Essentially all show aggressive clinical behavior independent of histologic grade and most are treated by mastectomy. MYC amplification is frequently identified in radiation-induced AS but only rarely in primary mammary AS (PMAS). As a heterogeneous group, AS from various anatomic sites have been shown to harbor recurrent alterations in TP53, MAP kinase pathway genes, and genes involved in angiogenic signaling including KDR (VEGFR2) and PTPRB. In part due to its rarity, the pathogenesis of PMAS has not been fully characterized. In this study, we examined the clinical, pathologic, and genomic features of ten cases of PMAS, including one patient with bilateral disease. Recurrent genomic alterations were identified in KDR (70%), PIK3CA/PIK3R1 (70%), and PTPRB (30%), each at higher frequencies than reported in AS across all sites. Six tumors harbored a KDR p.T771R hotspot mutation, and all seven KDR-mutant cases showed evidence suggestive of biallelism (four with loss of heterozygosity and three with two aberrations). Of the seven tumors with PI3K alterations, six harbored pathogenic mutations other than in the canonical PIK3CA residues which aremost frequent in breast cancer. Three AS were hypermutated (≥10 mutations/megabase (Mb)); hypermutation was seen concurrent with KDR or PIK3CA mutations. The patient with bilateral disease demonstrated shared alterations, indicative of contralateral metastasis. No MYC or TP53 aberrations were detected in this series. Immunohistochemistry for VEGFR2 was unable to discriminate between KDR-mutant tumors and benign vascular lesions of the breast. These findings highlight the underrecognized frequency of KDR and PIK3CA mutation in PMAS, and a significant subset with hypermutation, suggesting a pathogenesis distinct from other AS.

Effects of indoor residual spraying and outdoor larval control on Anopheles coluzzii from S\xe3o Tom\xe9 and Pr\xedncipe, two islands with pre-eliminated malaria

BackgroundVector control is a key component of malaria prevention. Two major vector control strategies have been implemented in São Tomé and Príncipe (STP), indoor residual spraying (IRS) and outdoor larval control using Bacillus thuringiensis israelensis (Bti). This study evaluated post-intervention effects of control strategies on vector population density, composition, and knockdown resistance mutation, and their implications for malaria epidemiology in STP.MethodsMosquitoes were collected by indoor and outdoor human landing catches and mosquito light traps in seven districts. Mosquito density was calculated by numbers of captured adult mosquitoes/house/working hour. Mitochondrial cytochrome c oxidase subunit I (COI) was PCR amplified and sequenced to understand the spatial–temporal population composition of malaria vector in STP. Knockdown resistance L1014F mutation was detected using allele-specific PCR. To estimate the malaria transmission risks in STP, a negative binomial regression model was constructed. The response variable was monthly incidence, and the explanatory variables were area, rainfall, entomological inoculation rate (EIR), and kdr mutation frequency.ResultsMalaria vector in STP is exophilic Anopheles coluzzii with significant population differentiation between Príncipe and São Tomé (mean FST = 0.16, p < 0.001). Both vector genetic diversity and knockdown resistance mutation were relatively low in Príncipe (mean of kdr frequency = 15.82%) compared to São Tomé (mean of kdr frequency = 44.77%). Annual malaria incidence rate in STP had been rapidly controlled from 37 to 2.1% by three rounds of country-wide IRS from 2004 to 2007. Long-term application of Bti since 2007 kept the mosquito density under 10 mosquitoes/house/hr/month, and malaria incidence rate under 5% after 2008, except for a rising that occurred in 2012 (incidence rate = 6.9%). Risk factors of area (São Tomé compared to Príncipe), rainfall, outdoor EIR, and kdr mutation frequency could significantly increase malaria incidence by 9.33–11.50, 1.25, 1.07, and 1.06 fold, respectively.ConclusionsIndoor residual spraying could rapidly decrease Anopheles density and malaria incidence in STP. Outdoor larval control using Bti is a sustainable approach for controlling local vector with exophilic feature and insecticide resistance problem. Vector control interventions should be intensified especially at the north-eastern part of São Tomé to minimize impacts of outbreaks.

Spatio-seasonal distribution of &lt;i&gt;Anopheles gambiae sensu lato&lt;/i&gt; and dynamics of the Voltage gate sodium channel knock down resistance mutation (&lt;i&gt;Vgsc1014F&lt;/i&gt;) in the city of Lomé, Togo

Poor planned urbanization sustains the presence of malaria vectors in some urban areas. Here, we provide information on the spatial and seasonal distribution of Anopheles gambiae sensu lato and the dynamics of kdr mutation in Lome, the capital city of Togo. Anopheles Larvae were collected in Lome from January to December 2005, to assess their frequency and abundance. Susceptibility tests were performed from 2006 to 2009 on adults of Anopheles gambiae s.l. that emerged from the larvae collected at Akodessewa following the WHO standard protocol, using DDT, deltamethrin, and permethrin. A molecular assessment of pyrethroid knockdown resistance ( Vgsc-1014F kdr) was also conducted. In 2005, a total of 2,397 Anopheles gambiae s.l. larvae were collected. Out of 124 adults identified by PCR, 92.7% were An. coluzzii/An. gambiae and 13.2% were An. arabiensis. Anopheles coluzzii was more frequent throughout the year regardless the season whereas An. gambiae and An. arabiensis were more specific to the rainy seasons. A decrease of susceptibility was observed in An. gambiae s.l. during the 3 rounds. The Vgsc-1014F kdr frequency varied from 7.5% to 8.7%. This situation could affect the success of the integrated distribution campaign to provide individuals with ITNs and LLINs in Togo. Keywords : Breeding sites, Anopheles gambiae s.l., insecticide resistance, dynamics, Vgsc-1014F kdr mutation.

Low Knockdown Resistance Frequency In Anopheles Gambiae Complex Resistant To Some Who-Approved Insecticides In Selected Areas Of Lagos State, South-West, Nigeria

Background: Decline in malaria prevalence is usually attributed to the efficient vector control strategies implemented in such endemic areas. The spread of insecticide resistance in Anopheles mosquitoes is a major drawback to the gains in malaria vector control. Here we assessed the susceptibility status of Anopheles gambiae s.l. to some selected WHO-approved insecticides and frequency of knockdown resistance gene in two Local Government Areas of Lagos State, Nigeria. Methods: Three to five days old adult female mosquitoes were exposed to deltamethrin, permethrin and bendocarb. Knockdown time was recorded every ten minutes and % mortality taken at 24hr post exposure. KDT50 and KDT95 were determined using probit regression analysis. The resistant mosquitoes were used for species identification by Polymerase Chain Reaction (PCR) assays and characterized for the knockdown resistance (kdr) mutation by allele-specific PCR (AS-PCR). Results: The results of twenty-four hour post exposure mortality rate showed that mosquitoes sampled at differ-ent locations were resistant to permethrin (39% mortality for Lagos Mainland L.G.A.; 45% for Kosofe L.G.A.), del-tamethrin (51% for Lagos Mainland L.G.A; 68% for Kosofe L.G.A), but susceptible to bendiocarb (100% for Lagos Mainland L.G.A.; 99% for Kosofe L.G.A.). The KDT50 varied from 34 minutes in bendiocarb for both LGAs to 190 minutes in deltamethrin for Kosofe and 119 minutes in permethrin for Lagos Mainland. In all insecticides tested for both LGAs, KDT95 was greater than 60 minutes. Only the 1014F kdr mutation was detected. The overall kdr frequen-cy was low (1.79%). There was no significant association between the presence of the 1014F kdr allele and ability of Anopheles mosquitoes to survive exposure to the tested insecticides (P &gt; 0.05). Conclusion: This level of resistance to WHO approved insecticides is a threat to control programs and can have significant operational impact, if action is not taken.

Frequency of kdr and kdr-his Alleles in Stable Fly (Diptera: Muscidae) Populations From the United States, Costa Rica, France, and Thailand.

Anecdotal evidence of pyrethroid insecticide product failure for the control of stable fly [Stomoxys calcitrans (L.)] populations in the United States and worldwide prompted us to evaluate the frequency of knockdown resistance (kdr)-type polymorphisms within the voltage-sensitive sodium channel (Vssc) gene of field collected specimens from the United States, France, Costa Rica, and Thailand. The kdr-his allele (L1014H), associated with permethrin resistance, was detected in stable flies from the 10 states sampled in the United States, as well as from Costa Rica and France (Toulouse). Field collections of stable flies from California (Modesto) and New York (Cliffton Springs) exhibited reduced susceptibility upon exposure to a diagnostic permethrin concentration of 10× LC99, but survival did not appear to strictly associate with frequency of the kdr-his allele. This suggests that there are additional resistance mechanisms contributing to the phenotype in these states. The kdr allele (L1014F) was detected for the first time in stable flies originating in France and Thailand, and an improved, DNA-based diagnostic assay was developed and validated for use in future screens for kdr and kdr-his allele frequencies from field collections. The absence of kdr in United States and Costa Rica populations suggests that the allele is currently restricted to Europe and Asia.

Target-site mutations (AChE-G119S and kdr) in Guangxi Anopheles sinensis populations along the China-Vietnam border

BackgroundIn South Asia, the epidemiology of malaria is complex, and transmission mainly occurs in remote areas near international borders. Vector control has been implemented as a key strategy in malaria prevention for decades. A rising threat to the efficacy of vector control efforts is the development of insecticide resistance, thus it is important to monitor the type and frequency of insecticide resistant alleles in the disease vectors such as An. sinensis along the China-Vietnam border. Such information is needed to synthesize effective malaria vector control strategies.MethodsA total of 208 adults of An. sinensis, collected from seven sites in southwest Guangxi along the China-Vietnam border, were inspected for the resistance-conferring G119S mutation in acetylcholinesterase (AChE) by PCR-RFLP (polymerase chain reaction restriction fragment length polymorphism) and kdr mutations in the voltage-gated sodium channel (VGSC) by sequencing. In addition, the evolutionary origin of An. sinensis vgsc gene haplotypes was analyzed using Network 5.0.ResultsThe frequencies of mutant 119S of AChE were between 0.61–0.85 in the seven An. sinensis populations. No susceptible homozygote (119GG) was detected in three of the seven sites (DXEC, LZSK and FCGDX). Very low frequencies of kdr (0.00–0.01) were detected in the seven populations, with most individuals being susceptible homozygote (1014LL). The 1014F mutation was detected only in the southeast part (FCGDX) at a low frequency of 0.03. The 1014S mutation was distributed in six of the seven populations with frequencies ranging from 0.04 to 0.08, but absent in JXXW. Diverse haplotypes of 1014L and 1014S were found in An. sinensis along the China-Vietnam border, while only one 1014F haplotype was detected in this study. Consistent with a previous report, resistant 1014S haplotypes did not have a single origin.ConclusionsThe G119S mutation of AChE was present at high frequencies (0.61–0.85) in the An. sinensis populations along the China-Vietnam border, suggesting that the vector control authorities should be cautious when considering carbamates and organophosphates as chemicals for vector control. The low frequencies (0.00–0.11) of kdr in these populations suggest that pyrethroids remain suitable for use against An. sinensis in these regions.

Insecticide Resistance Status and Mechanisms of Anopheles sinensis (Diptera: Culicidae) in Wenzhou, an Important Coastal Port City in China.

Although scaled-up interventions and effective control efforts have drastically reduced malaria morbidity and mortality, malaria remains a serious threat to public health worldwide. Anopheles sinensis Wiedemann 1828 is a historically important vector of Plasmodium vivax (Haemosporida: Plasmodiidae) malaria in China. Insecticide resistance has become a major obstacle to vector-borne disease control. However, little is known about the insecticide resistance of An. sinensis in Wenzhou, an important coastal port city in Zhejiang province, China. The aim of this study was to examine insecticide resistance and mechanisms in An. sinensis field mosquito populations. Evidence of multiple insecticide resistance was found in An. sinensis adult female populations. Medium to high frequencies of target site kdr together with fixed ace-1 mutations was detected in both the Ruian and Yongjia populations. Both populations showed an association between kdr L1014 mutation and resistance phenotype when tested against deltamethrin and DDT. Significantly different metabolic enzyme activities were found between the susceptible laboratory strain and field-collected mosquitoes from both Ruian and Yongjia. Both field collected An. sinensis populations exhibited significantly higher P450 enzyme activity compared with the laboratory strain, while the field-collected resistant mosquitoes exhibited various GST and COE enzyme activities. These results indicate multiple resistance mechanisms in An. sinensis field populations. Effective implementation of insecticide resistance management strategies is urgently needed. The data collected in this study will be valuable for modeling insecticide resistance spread and vector-control interventions.

Insecticide resistance status, frequency of L1014F Kdr and G119S Ace-1 mutations, and expression of detoxification enzymes in Anopheles gambiae (s.l.) in two regions of northern Benin in preparation for indoor residual spraying

BackgroundThis study aims to provide baseline data on the resistance status to insecticides, the frequency of mechanisms involved and the impact of the association with the synergist piperonyl butoxide (PBO) on resistant Anopheles gambiae (s.l.) populations in two regions of northern Benin, prior to an indoor residual spraying campaign and introduction of next generation long-lasting insecticidal nets (LLINs) incorporating PBO.MethodsAdult Anopheles gambiae (s.l.) originating from larvae collected in two study regions (Alibori within the Kandi-Gogounou-Segbana districts and Donga within the Djougou-Copargo-Ouake districts) were tested with impregnated papers (bendiocarb 0.1%, pirimiphos-methyl 0.25%, permethrin 0.75% and deltamethrin 0.05%). The synergist PBO was used to check for the involvement of detoxification enzymes in pyrethroid resistant populations. Molecular analyses were performed for the identification of species within the Anopheles gambiae (s.l.) complex and kdr L1014F and G119S Ace-1 mutations. Biochemical assays assessed the activity of detoxification enzymes.ResultsAnopheles gambiae (s.l.) was resistant to pyrethroids, with a mortality range of 25–83% with deltamethrin and 6–55% with permethrin. A significant increase in mortality was observed after pre-exposure to PBO for both deltamethrin (63–99%) and permethrin (56–99%). With bendiocarb, An. gambiae (s.l.) were susceptible in Kandi (99% mortality), with possible resistance (92–95%) recorded in Djougou, Copargo, Gogounou, Ouake and Segbana. All study populations were fully susceptible to pirimiphos-methyl. The frequencies of resistant mutations varied according to species and sites: 0.67–0.88 for L1014F kdr and 0–0.06 for G119S Ace-1. Three study locations (Djougou, Gogounou and Kandi) showed high oxidase activity and four sites (Djougou, Ouake, Copargo and Kandi) showed elevated esterase activity.ConclusionsThis study confirms resistance to pyrethroids and suggests emerging bendiocarb resistance in An. gambiae (s.l.) populations in northern Benin. However, recovery of susceptibility to pyrethroids after PBO exposure, and susceptibility to organophosphates in the An. gambiae (s.l.) populations indicate that next generation LLINs incorporating PBO synergist combined with an indoor residual spraying (IRS) campaign with organophosphate insecticides may be regarded as alternative control tools.

Multiple insecticide resistance in Culex quinquefasciatus populations from Guadeloupe (French West Indies) and associated mechanisms.

West Nile (WN) virus has been detected in Guadeloupe since 2002. Even if no WN human cases have been detected so far, mosquitoes from Culex genus especially Culex quinquefasciatus are recognized as potential WN vectors in Guadeloupe. To evaluate the impact of local vector control activities on this mosquito species we assessed the resistance levels of Cx. quinquefasciatus populations from three different sites from Guadeloupe (Abymes, Saint François and Gourbeyre) to malathion, temephos and deltamethrin. In addition, the frequencies of the L1014F kdr and the G119S ace-1 mutations were established in Cx. quinquefasciatus populations, as well as the constitutive expressions of five cytochrome P450 genes. Mosquito populations tested displayed high resistance to deltamethrin, moderate resistance to malathion (Abymes, Gourbeyre) and low resistance to temephos (Abymes et Gourbeyre). Molecular analyses revealed high frequencies of both L1014F kdr and G119S ace-1 mutations in Cx. quinquefasciatus populations, as well as overexpression of cytochrome P450 genes CYP9J45, CYP9J40 and CYP6AA7. Finally, deltamethrin resistance and knock-down rates were strongly correlated with the frequency of the resistant kdr and ace-1 alleles, as well as with CYP9J40 overexpression. These results should be taken into account to refine the current vector control strategies to prevent the appearance of Cx. quinquefasciatus-borne diseases in Guadeloupe.

Rapid selection of a pyrethroid metabolic enzyme CYP9K1 by operational malaria control activities.

Since 2004, indoor residual spraying (IRS) and long-lasting insecticide-impregnated bednets (LLINs) have reduced the malaria parasite prevalence in children on Bioko Island, Equatorial Guinea, from 45% to 12%. After target site-based (knockdown resistance; kdr) pyrethroid resistance was detected in 2004 in Anopheles coluzzii (formerly known as the M form of the Anopheles gambiae complex), the carbamate bendiocarb was introduced. Subsequent analysis showed that kdr alone was not operationally significant, so pyrethroid-based IRS was successfully reintroduced in 2012. In 2007 and 2014-2015, mass distribution of new pyrethroid LLINs was undertaken to increase the net coverage levels. The combined selection pressure of IRS and LLINs resulted in an increase in the frequency of pyrethroid resistance in 2015. In addition to a significant increase in kdr frequency, an additional metabolic pyrethroid resistance mechanism had been selected. Increased metabolism of the pyrethroid deltamethrin was linked with up-regulation of the cytochrome P450 CYP9K1. The increase in resistance prompted a reversion to bendiocarb IRS in 2016 to avoid a resurgence of malaria, in line with the national Malaria Control Program plan.

Targeted application of an organophosphate-based paint applied on windows and doors against Anopheles coluzzii resistant to pyrethroids under real life conditions in Vall\xe9e du Kou, Burkina Faso (West Africa)

BackgroundA novel strategy applying an organophosphate-based insecticide paint on doors and windows in combination with long-lasting insecticide-treated nets (LLINs) was tested for the control of pyrethroid-resistant malaria vectors in a village setting in Vallée du Kou, a rice-growing area west of Burkina Faso.MethodsInsecticide Paint Inesfly 5A IGR™, comprised of two organophosphates and an insect growth regulator, was applied to doors and windows and tested in combination with pyrethroid-treated LLINs. The killing effect was monitored for 5 months by early morning collections of anophelines and other culicids. The residual efficacy was evaluated monthly by WHO bioassays using Anopheles gambiae ‘Kisumu’ and local populations of Anopheles coluzzii resistant to pyrethroids. The spatial mortality efficacy (SME) at distances of 1 m was also assessed against pyrethroid-susceptible and -resistant malaria vectors. The frequency of L1014F kdr and Ace-1R G119S mutations was, respectively, reported throughout the study. The Insecticide Paint Inesfly 5A IGR had been tested in past studies yielding a long-term mortality rate of 80% over 12 months against An. coluzzii, the local pyrethroid-resistant malaria vector. The purpose of the present study is to test if treating smaller, targeted surfaces (e.g. doors and windows) was also efficient in killing malaria vectors.ResultsTreating windows and doors alone yielded a killing efficacy of 100% for 1 month against An. coluzzii resistant to pyrethroids, but efficacy reduced quickly afterwards. Likewise, WHO cone bioassays yielded mortalities of 80–100% for 2 months but declined to 90 and 40% 2 and 3 months after treatment, respectively. Mosquitoes exposed to insecticide paint-treated surfaces at distances of 1 m, yielded mortality rates of about 90–80% against local pyrethroids-resistant An. coluzzii during the first 2 months, but decreased to 30% afterwards. Anopheles coluzzii was reported to be exclusively the local malaria vector and resistant to pyrethroids with high L1014 kdr frequency.ConclusionThe combination of insecticide paint on doors and windows with LLINs yielded high mortality rates in the short term against wild pyrethroid-resistant malaria vector populations. A high SME was observed against laboratory strains of pyrethroid-resistant malaria vectors placed for 30 min at 1 m from the treated/control walls. The application of the insecticide paint on doors and windows led to high but short-lasting mortality rates. The strategy may be an option in a context where low cost, rapid responses need to be implemented in areas where malaria vectors are resistant to pyrethroids.

Status of insecticide resistance in malaria vectors in Kwale County, Coastal Kenya

BackgroundThe strategy for malaria vector control in the context of reducing malaria morbidity and mortality has been the scale-up of long-lasting insecticidal nets to universal coverage and indoor residual spraying. This has led to significant decline in malaria transmission. However, these vector control strategies rely on insecticides which are threatened by insecticide resistance. In this study the status of pyrethroid resistance in malaria vectors and it’s implication in malaria transmission at the Kenyan Coast was investigated.ResultsUsing World Health Organization diagnostic bioassay, levels of phenotypic resistance to permethrin and deltamethrin was determined. Anopheles arabiensis showed high resistance to pyrethroids while Anopheles gambiae sensu stricto (s.s.) and Anopheles funestus showed low resistance and susceptibility, respectively. Anopheles gambiae sensu lato (s.l.) mosquitoes were further genotyped for L1014S and L1014F kdr mutation by real time PCR. An allele frequency of 1.33% for L1014S with no L1014F was detected. To evaluate the implication of pyrethroid resistance on malaria transmission, Plasmodium falciparum infection rates in field collected adult mosquitoes was determined using enzyme linked immunosorbent assay and further, the behaviour of the vectors was assessed by comparing indoor and outdoor proportions of mosquitoes collected. Sporozoite infection rate was observed at 4.94 and 2.60% in An. funestus s.l. and An. gambiae s.l., respectively. A higher density of malaria vectors was collected outdoor and this also corresponded with high Plasmodium infection rates outdoor.ConclusionsThis study showed phenotypic resistance to pyrethroids and low frequency of L1014S kdr mutation in An. gambiae s.l. The occurrence of phenotypic resistance with low levels of kdr frequencies highlights the need to investigate other mechanisms of resistance. Despite being susceptible to pyrethroids An. funestus s.l. could be driving malaria infections in the area.

Insecticide resistance patterns in Uganda and the effect of indoor residual spraying with bendiocarb on kdr L1014S frequencies in Anopheles gambiae s.s.

BackgroundResistance of malaria vectors to pyrethroid insecticides has been attributed to selection pressure from long-lasting insecticidal nets (LLINs), indoor residual spraying (IRS), and the use of chemicals in agriculture. The use of different classes of insecticides in combination or by rotation has been recommended for resistance management. The aim of this study was to understand the role of IRS with a carbamate insecticide in management of pyrethroid resistance.MethodsAnopheles mosquitoes were collected from multiple sites in nine districts of Uganda (up to five sites per district). Three districts had been sprayed with bendiocarb. Phenotypic resistance was determined using standard susceptibility tests. Molecular assays were used to determine the frequency of resistance mutations. The kdr L1014S homozygote frequency in Anopheles gambiae s.s. was used as the outcome measure to test the effects of various factors using a logistic regression model. Bendiocarb coverage, annual rainfall, altitude, mosquito collection method, LLIN use, LLINs distributed in the previous 5 years, household use of agricultural pesticides, and malaria prevalence in children 2–9 years old were entered as explanatory variables.ResultsTests with pyrethroid insecticides showed resistance and suspected resistance levels in all districts except Apac (a sprayed district). Bendiocarb resistance was not detected in sprayed sites, but was confirmed in one unsprayed site (Soroti). Anopheles gambiae s.s. collected from areas sprayed with bendiocarb had significantly less kdr homozygosity than those collected from unsprayed areas. Mosquitoes collected indoors as adults had significantly higher frequency of kdr homozygotes than mosquitoes collected as larvae, possibly indicating selective sampling of resistant adults, presumably due to exposure to insecticides inside houses that would disproportionately affect susceptible mosquitoes. The effect of LLIN use on kdr homozygosity was significantly modified by annual rainfall. In areas receiving high rainfall, LLIN use was associated with increased kdr homozygosity and this association weakened as rainfall decreased, indicating more frequency of exposure to pyrethroids in relatively wet areas with high vector density.ConclusionThis study suggests that using a carbamate insecticide for IRS in areas with high levels of pyrethroid resistance may reduce kdr frequencies in An. gambiae s.s.

Knockdown resistance mutations predict DDT resistance and pyrethroid tolerance in the visceral leishmaniasis vector Phlebotomus argentipes.

BackgroundIndoor residual spraying (IRS) with DDT has been the primary strategy for control of the visceral leishmaniasis (VL) vector Phlebotomus argentipes in India but efficacy may be compromised by resistance. Synthetic pyrethroids are now being introduced for IRS, but with a shared target site, the para voltage-gated sodium channel (VGSC), mutations affecting both insecticide classes could provide cross-resistance and represent a threat to sustainable IRS-based disease control.Methodology/Principal findingsA region of the Vgsc gene was sequenced in P. argentipes from the VL hotspot of Bihar, India. Two knockdown resistance (kdr) mutations were detected at codon 1014 (L1014F and L1014S), each common in mosquitoes, but previously unknown in phlebotomines. Both kdr mutations appear largely recessive, but as homozygotes (especially 1014F/F) or as 1014F/S heterozygotes exert a strong effect on DDT resistance, and significantly predict survivorship to class II pyrethroids in short-duration bioassays. The mutations are present at high frequency in wild P. argentipes populations from Bihar, with 1014F significantly more common in higher VL areas.Conclusions/SignificanceThe Vgsc mutations detected appear to be a primary mechanism underlying DDT resistance in P. argentipes and a contributory factor in reduced pyrethroid susceptibility, suggesting a potential impact if P. argentipes are subjected to suboptimal levels of pyrethroid exposure, or additional resistance mechanisms evolve. The assays to detect kdr frequency changes provide a sensitive, high-throughput monitoring tool to detecting spatial and temporal variation in resistance in P. argentipes.

A neural network prediction of environmental determinants of Anopheles sinensis knockdown resistance mutation to pyrethroids in China.

Selection pressure caused by long-term intensive use of insecticides is the key driving force in resistance development. Additional parameters such as environmental conditions may affect both the mosquito response to insecticides and the selection of resistance mechanisms. In this context, we analyzed the environmental determinants of kdr prevalence in Anopheles sinensis across China. We collected kdr frequency from 48 sites across central and southern China, together with key environmental factors including long-term climatic data, topographic features, main crops, and land cover types. Trend surface analysis found that the distribution of kdr frequency can be partitioned into three regions, namely central China (kdr frequency >80%), western China (kdr frequency varies from 0% to 60%), and southern China (kdr frequency <10%). Seven predictor variables were selected based on a radial basis function neural network model. A multilayer perceptron (MLP) network model revealed that the number of crops in a year was the most important predictor for the kdr mutation rate. Topography, long-term mean climate and land cover all contributed to the kdr mutation rate. The observed mean kdr frequency was 53.0% and the MLP network model-predicted mean was 52.6%, a 0.1% relative error. Predicted kdr frequencies closely matched the observed values. The model explained 92% of the total variance in kdr frequency. The results indicated that kdr was associated with the intensity of pesticide usage. Crop cultivation information, together with environmental factors, may well predict the spatial heterogeneity of kdr mutations in An. sinensis in China.

Multiple insecticide resistances and kdr mutation of Culex pipiens pallens in north-central Anhui Province

To understand the status of resistance to multiple insecticides and the frequencies of kdr mutations in Culex pipiens pallens from north-central Anhui Province. From July to September, 2014, the C. pipiens pallens mosquito larvae were collected in Huaibei, Bengbu and Chuzhou cities of the north-central Anhui Province and reared to adults. The female adult mosquitoes at 3-5 days post emergence were tested for susceptibility to the four insecticides, namely 0.05% deltamethrin, 5% malathion, 0.1% bendiocarb and 4% DDT, by using the standard WHO resistance tube bioassay. The detection of the point mutations of the kdr gene at codon 1014 was conducted by PCR and DNA sequencing in the deltamethrin-resistant and -susceptible mosquitoes. High levels of resistance to all the four insecticides were found in all the three tested populations, although mosquito mortality varied among populations and test insecticides. Among the test insecticides, DDT showed lowest mortality with no significant difference (F = 1.027, P > 0.05) in all test populations, whereas significantly different mortalities were observed among populations for the remained three insecticides tested (deltamethrin, malathion, and bendiocarb) (F = 23.823, 33.955, 128.841; all P < 0.01). Two types of non-synonymous kdr mutation at codon position 1014 (L1014F and L1014S) were observed. A positive correlation between L1014F mutation frequencies and deltamethrin resistance levels were detected in the three mosquito populations (r2 = 0.718, P < 0.01). The observed high levels of resistance to multiple-insecticides coupled with the occurrence of medium to high kdr frequencies in populations of C. pipiens pallens could profoundly affect the mosquito vector control programme in China. The local health departments need to strengthen vector dynamic monitoring and implement rational resistance management strategies.

Insecticide susceptibility of natural populations of Anopheles coluzzii and Anopheles gambiae (sensu stricto) from Okyereko irrigation site, Ghana, West Africa.

BackgroundThe increasing spread of insecticide resistance in malaria vectors has been well documented across sub-Saharan Africa countries. The influence of irrigation on increasing vector resistance is poorly understood, and is critical to successful and ethical implementation of food security policies. This study investigated the insecticide resistance status of An. gambiae (s.l.) mosquitoes collected from the irrigated rice area of Okyereko, a village containing about 42 hectares of irrigated field within an irrigation project plan in the Central Region of Ghana. Large amounts of insecticides, herbicides and fertilizers are commonly used in the area to boost the annual production of the rice.MethodsMosquito larvae were collected and adults were assayed from the F1 progeny. The resistance status, allele and genotype were characterized using WHO susceptibility testing and PCR methods respectively.ResultsThe An. gambiae (s.l.) populations from Okyereko are highly resistant to DDT and pyrethroid insecticides, with possible involvement of metabolic mechanisms including the elevation of P450 and GST enzyme as well as P-gp activity. The population was mostly composed of An. coluzzii specimens (more than 96 %) with kdr and ace-1 frequencies of 0.9 and 0.2 %, respectively.ConclusionThis study brings additional information on insecticide resistance and the characterization of An. gambiae (s.l.) mosquitoes from Okyereko, which can be helpful in decision making for vector control programmes in the region.

Phenotypic and genotypic profile of pyrethroid resistance in populations of the mosquito Aedes aegypti from Goiânia, Central West Brazil.

The mosquito Aedes aegypti has evolved resistance to pyrethroid insecticides. The present study evaluated Ae. aegypti from Goiânia for the resistant phenotype and for mutations associated with resistance. Insecticide dose-response bioassays were conducted on mosquitoes descended from field-collected eggs, and polymerase chain reaction (PCR) was used to genotype 90 individuals at sites implicated in pyrethroid resistance. All mosquito populations displayed high levels of resistance to deltamethrin, as well as high frequencies of the 1016Ile kdr and 1534Cys kdrmutations. Aedes aegypti populations in the Goiânia area are highly resistant to deltamethrin, presumably due to high frequencies of kdr(knockdown-resistance) mutations.

Characterizing the insecticide resistance of Anopheles gambiae in Mali.

BackgroundThe impact of indoor residual spraying (IRS) and long-lasting insecticide nets (LLINs), key components of the national malaria control strategy of Mali, is threatened by vector insecticide resistance. The objective of this study was to assess the level of insecticide resistance in Anopheles gambiae sensu lato populations from Mali against four classes of insecticide recommended for IRS: organochlorines (OCs), pyrethroids (PYs), carbamates (CAs) and organophosphates (OPs). Characterization of resistance was done in 13 sites across southern Mali and assessed presence and distribution of physiological mechanisms that included target-site modifications: knockdown resistance (kdr) and altered acetycholinesterase (AChE), and/or metabolic mechanisms: elevated esterases, glutathione S-transferases (GSTs), and monooxygenases.MethodsThe World Health Organization (WHO) tube test was used to determine phenotypic resistance of An.gambiae s.l. to: dichlorodiphenyltrichloroethane (DDT) (OC), deltamethrin (PY), lambda-cyhalothrin (PY), bendiocarb (CA), and fenitrothion (OP). Identification of sibling species and presence of the ace-1R and Leu-Phe kdr, resistance-associated mutations, were determined using polymerase chain reaction (PCR) technology. Biochemical assays were conducted to detect increased activity of GSTs, oxidases and esterases.ResultsPopulations tested showed high levels of resistance to DDT in all 13 sites, as well as increased resistance to deltamethrin and lambda-cyhalothrin in 12 out of 13 sites. Resistance to fenitrothion and bendiocarb was detected in 1 and 4 out of 13 sites, respectively. Anopheles coluzzii, An. gambiae sensu stricto and Anopheles arabiensis were identified with high allelic frequencies of kdr in all sites where each of the species were found (13, 12 and 10 sites, respectively). Relatively low allelic frequencies of ace-1R were detected in four sites where this assessment was conducted. Evidence of elevated insecticide metabolism, based on oxidase, GSTs and esterase detoxification, was also documented.ConclusionMultiple insecticide-resistance mechanisms have evolved in An. coluzzii, An. gambiae s.s. and An.arabiensis in Mali. These include at least two target site modifications: kdr, and ace-1R, as well as elevated metabolic detoxification systems (monooxygenases and esterases). The selection pressure for resistance could have risen from the use of these insecticides in agriculture, as well as in public health. Resistance management strategies, based on routine resistance monitoring to inform insecticide-based malaria vector control in Mali, are recommended.

Impact of long-lasting, insecticidal nets on anaemia and prevalence of Plasmodium falciparum among children under five years in areas with highly resistant malaria vectors

BackgroundThe widespread use of insecticide-treated nets (LLINs) leads to the development of vector resistance to insecticide. This resistance can reduce the effectiveness of LLIN-based interventions and perhaps reverse progress in reducing malaria morbidity. To prevent such difficulty, it is important to know the real impact of resistance in the effectiveness of mosquito nets. Therefore, an assessment of LLIN efficacy was conducted in malaria prevention among children in high and low resistance areas.MethodsThe study was conducted in four rural districts and included 32 villages categorized as low or high resistance areas in Plateau Department, south-western Benin. Larvae collection was conducted to measure vector susceptibility to deltamethrin and knockdown resistance (kdr) frequency. In each resistance area, around 500 children were selected to measure the prevalence of malaria infection as well as the prevalence of anaemia associated with the use of LLINs.ResultsObserved mortalities of Anopheles gambiae s.s population exposed to deltamethrin ranged from 19 to 96%. Knockdown resistance frequency was between 38 and 84%. The prevalence of malaria infection in children under five years was 22.4% (19.9-25.1). This prevalence was 17.3% (14.2-20.9) in areas of high resistance and 27.1% (23.5-31.1) in areas of low resistance (p = 0.04). Eight on ten children that were aged six - 30 months against seven on ten of those aged 31–59 months were anaemic. The anaemia observed in the six to 30-month old children was significantly higher than in the 31–59 month old children (p = 0.00) but no difference associated with resistance areas was observed (p = 0.35). The net use rate was 71%. The risk of having malaria was significantly reduced (p < 0.05) with LLIN use in both low and high resistance areas. The preventive effect of LLINs in high resistance areas was 60% (95% CI: 40–70), and was significantly higher than that observed in low resistance areas (p < 0.05).ConclusionThe results of this study showed that the resistance of malaria vectors seems to date not have affected the impact of LLINs and the use of LLINs was highly associated with reduced malaria prevalence irrespective of resistance.