Longitudinal survey of insecticide resistance in a village of central region of Burkina Faso reveals co-occurrence of 1014F, 1014S and 402L mutations in Anopheles coluzzii and Anopheles arabiensis.

BACKGROUND: Pyrethroid resistance is one of the major threats for effectiveness of insecticide-treated bed nets (ITNs) in malaria vector control. Genotyping of mutations in the voltage gated sodium channel (VGSC) gene is widely used to easily assess the evolution and spread of pyrethroid target-site resistance among malaria vectors. L1014F and L1014S substitutions are the most common and best characterized VGSC mutations in major African malaria vector species of the Anopheles gambiae complex. Recently, an additional substitution involved in pyrethroid resistance, i.e. V402L, has been detected in Anopheles coluzzii from West Africa lacking any other resistance alleles at locus 1014. The evolution of target-site resistance mutations L1014F/S and V402L was monitored in An. coluzzii and Anopheles arabiensis specimens from a Burkina Faso village over a 10-year range after the massive ITN scale-up started in 2010. METHODS: Anopheles coluzzii (N = 300) and An. arabiensis (N = 362) specimens collected both indoors and outdoors by different methods (pyrethrum spray catch, sticky resting box and human landing collections) in 2011, 2015 and 2020 at Goden village were genotyped by TaqMan assays and sequencing for the three target site resistance mutations; allele frequencies were statistically investigated over the years. RESULTS: A divergent trend in resistant allele frequencies was observed in the two species: 1014F decreased in An. coluzzii (from 0.76 to 0.52) but increased in An. arabiensis (from 0.18 to 0.70); 1014S occurred only in An. arabiensis and slightly decreased over time (from 0.33 to 0.23); 402L increased in An. coluzzii (from 0.15 to 0.48) and was found for the first time in one An. arabiensis specimen. In 2020 the co-occurrence of different resistance alleles reached 43% in An. coluzzii (alleles 410L and 1014F) and 32% in An. arabiensis (alleles 1014F and 1014S). CONCLUSIONS: Overall, an increasing level of target-site resistance was observed among the populations with only 1% of the two malaria vector species being wild type at both loci, 1014 and 402, in 2020. This, together with the co-occurrence of different mutations in the same specimens, calls for future investigations on the possible synergism between resistance alleles and their phenotype to implement local tailored intervention strategies.

Impact of a spatial repellent intervention on Anopheles kdr insecticide resistance allele in Sumba, Indonesia.

BACKGROUND: The emergence of insecticide resistance and outdoor transmission in malaria-endemic areas underlines the urgent need to develop innovative tools, such as spatial repellents (SR), that may circumvent this residual transmission. With limited options for effective insecticides, regular resistance monitoring is warranted for selecting and using appropriate tools. This study evaluates the pyrethroid knockdown resistance (kdr) allele before and after implementing a transfluthrin-based spatial repellent (SR) intervention in placebo-treated clusters. METHODS: This study looks at the frequency distribution of the kdr allele in Sumba Island from June 2015 to August 2018. Insecticide susceptibility tests were carried out on female Anopheles sp. aged 3-5 days against permethrin 21.5 µg/ml, deltamethrin 12.5 µg/ml, and transfluthrin 10 µg/ml using CDC bottle assay. PCR sequencing of representative samples from adult mosquito collections and insecticide tests revealed the presence of kdr mutations (L1014F and L1014S) in the VGSC gene. RESULTS: A total of 12 Anopheles species, Anopheles tesselatus, Anopheles. aconitus, Anopheles barbirostris, Anopheles kochi, Anopheles annularis, Anopheles maculatus, Anopheles sundaicus, Anopheles flavirostris, Anopheles balabacensis, Anopheles indefinitus, Anopheles subpictus, and Anopheles vagus were analysed. Anopheles vagus and An. sundaicus predominated in the larval populations. Susceptibility assays for all insecticides identified fully susceptible phenotypes in all species examined. Anopheles increasing frequency of kdr mutant alleles during the 3 year SR deployment was observed in both SR-treated and placebo areas, a statistically significant increase occurred in each arm. However, it is unclear how significant SR is in causing the increase in mutant alleles. The L1014S, knockdown resistance east type (kdr-e) allele was detected for the first time among the mosquito samples in this study. The L1014F, knockdown resistance west type (kdr-w) allele and heteroduplex form (wild-type-mutant) were found in almost all Anopheles species examined, including An. vagus, An. aconitus, An. subpictus, An. tesselatus, An. annularis, An. flavirostris and An. sundaicus. CONCLUSION: The presence of fully susceptible phenotypes over time, along with an increase in the frequency distribution of the L1014F/S mutations post-intervention, suggest drivers of resistance external to the study, including pyrethroid use in agriculture and long-lasting insecticidal nets (LLINs). However, this does not negate possible SR impacts that support resistance. More studies that enable the comprehension of possible SR-based drivers of resistance in mosquitoes need to be conducted.

Detection of Target Site Mutations in the Acetylcholinesterase and Voltage-Gated Sodium Channel in Field Populations of Culex quinquefasciatus and Cx. tritaeniorhynchus From Southern Sichuan Region of China.

Culex quinquefasciatus and Cx. tritaeniorhynchus are 2 dominant disease vectors in Neijiang City, Sichuan Province, China. Although there is evidence of confirmed resistance against insecticides in mosquito vectors, nothing is known about the existing insecticide resistance-conferring mutations in Cx. quinquefasciatus and Cx. tritaeniorhynchu in this region so far. In this study, the G119S mutation in the acetylcholinesterase (AChE) was detected in Cx. quinquefasciatus at a very low frequency (0.9%) with no resistant homozygotes being observed. Two resistance mutations in the voltage-gated sodium channel (VGSC) (L1014F and L1014S) were found in Cx. quinquefasciatus with frequencies of 88.7% and 8.3%, respectively. By contrast, the AChE F455W mutation was found to be fixed (with a frequency of 100%) in 3 of the 5 studied populations, with an overall frequency being 98.1%. In addition, 1 resistance-conferring VGSC mutation (L1014F) was detected with an overall frequency of 15.2% in Cx. tritaeniorhynchus. These results indicate that the well-recognized insecticide resistance-conferring mutations in both AChE and VGSC are present in the 2 Culex species in Neijiang. The contrasting patterns in the frequency of resistance alleles indicate that species-customized strategies of insecticide resistance management should be considered for the 2 species.

Trends in insecticide resistance in Culex pipiens pallens over 20 years in Shandong, China.

BACKGROUND: Culex pipiens pallens is the most abundant Culex mosquito species in northern China and is an important vector of bancroftian filariasis and, potentially, West Nile virus. Insecticides, particularly pyrethroids, are widely used for adult mosquito control. Insecticide resistance has become common in several mosquito species, and vector control is the main method currently available to prevent disease transmission. The voltage-gated sodium channel (Vgsc) gene is the target site of pyrethroids, and mutations in this gene cause knockdown resistance (kdr). METHODS: Culex pipiens pallens larvae were collected from May to November over two decades, from 1992 to 2018, in four cities in Shandong Province, China. The World Health Organization (WHO) standard resistance bioassay was applied to test the resistance levels of Cx. p. pallens larvae to five different insecticides and to test deltamethrin resistance in adults, using the F1 generation. Mutations at Vgsc codon 1014 were also screened in 471 adult samples collected in 2014 to determine the association between kdr mutations and phenotypic resistance. RESULTS: Larval resistance against deltamethrin showed an increasing trend from the 1990s until 2018, which was statistically significant in all populations; resistance to cypermethrin increased significantly in mosquitoes from the Zaozhuang population. However, larval resistance to other insecticides remained relatively stable. Larval resistance against deltamethrin was consistent with adult bioassays in 2014, in which all tested populations were highly resistant, with mortality rates ranging from 39.4 to 55.23%. The L1014S and L1014F mutations were both observed in five Cx. p. pallens populations, with L1014F significantly associated with deltamethrin resistance. CONCLUSIONS: The long-term dataset from Shandong demonstrates major increases in pyrethroid resistance over a 20-year period. The L1014F kdr mutation may be considered a viable molecular marker for monitoring pyrethroid resistance in Cx. p. pallens.

Detection of alleles associated with resistance to chemical insecticide in the malaria vector Anopheles arabiensis in Santiago, Cabo Verde.

BACKGROUND: Mosquitoes of the Anopheles gambiae complex are the main malaria vectors worldwide. Due to the lack of a vaccine to prevent malaria, the principal way to reduce the impact of this disease relies on the use of chemical insecticides to control its vectors. However, the intensive use of such compounds has led to the emergence of insecticide resistance in several Anopheles populations in Africa. This study aimed to investigate the presence of resistance alleles in an Anopheles arabiensis population from the City of Praia, capital of the Archipelago Cabo Verde, one of the countries on the World Health Organization list of countries that are on a path to eliminate local transmission of malaria. METHODS: Larvae from the Anopheles genus were collected using a one-pint dipper in three areas of City of Praia. Larvae were fed and maintained until the emergence of adult mosquitoes, and these were morphologically identified. In addition, molecular identification was performed using IGS markers and all An. arabiensis samples were subjected to PCR to screen for mutations associated to resistance in the Ace-1, Nav and GSTE2 genes. RESULTS: From a total of 440 mosquitoes collected, 52.3% were morphologically identified as An. gambiae sensu lato (s.l.) and 46.7% as Anopheles pretoriensis. The molecular identification showed that 100% of the An. gambiae s.l. were An. arabiensis. The mutations G119S in the Ace-1 gene and L119F in the GSTE2 gene were screened but not found in any sample. However, sequencing analysis for GSTE2 revealed the presence of 37 haplotypes, 16 polymorphic sites and a high genetic diversity (π = 2.67). The L1014S mutation in the Nav (voltage-gated sodium channel gene) was detected at a frequency of 7.3%. CONCLUSION: This is the first study to investigate the circulation of insecticide resistance alleles in An. arabiensis from Cabo Verde. The circulation of the L1014S allele in the population of An. arabiensis in the city of Praia suggests that pyrethroid resistance may arise, be quickly selected, and may affect the process of malaria elimination in Cabo Verde. Molecular monitoring of resistance should continue in order to guide the development of strategies to be used in vector control in the study region.

Detection and Monitoring of Insecticide Resistance Mutations in Anopheles gambiae: Individual vs Pooled Specimens.

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 ΔCt 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.

Insecticide resistance in Anopheles arabiensis populations from Dakar and its suburbs: role of target site and metabolic resistance mechanisms.

BACKGROUND: Urban malaria is an increasing concern in most of the sub-Saharan Africa countries. In Dakar, the capital city of Senegal, the malaria epidemiology has been complicated by recurrent flooding since 2005. The main vector control measure for malaria prevention in Dakar is the community use of long-lasting insecticide-treated nets. However, the increase of insecticide resistance reported in this area needs to be better understood for suitable resistance management. This study reports the situation of insecticide resistance and underlying mechanisms in Anopheles arabiensis populations from Dakar and its suburbs. RESULTS: All the populations tested showed resistance to almost all insecticides except organophosphates families, which remain the only lethal molecules. Piperonil butoxide (PBO) and ethacrinic acid (EA) the two synergists used, have respectively and significantly restored the susceptibility to DDT and permethrin of Anopheles population. Molecular identification of specimens revealed the presence of An. arabiensis only. Kdr genotyping showed the presence of the L1014F mutation (kdr-West) as well as L1014S (kdr-East). This L1014S mutation was found at very high frequencies (89.53%) in almost all districts surveyed, and in association with the L1014F (10.24%). CONCLUSION: Results showed the contribution of both target-site and metabolic mechanisms in conferring pyrethroid resistance to An. arabiensis from the flooded areas of Dakar suburbs. These data, although preliminary, stress the need for close monitoring of the urban An. arabiensis populations for a suitable insecticide resistance management system to preserve core insecticide-based vector control tools in this flooded area.

A significant association between deltamethrin resistance, Plasmodium falciparum infection and the Vgsc-1014S resistance mutation in Anopheles gambiae highlights the epidemiological importance of resistance markers.

BACKGROUND: The success of malaria vector control is threatened by widespread pyrethroid insecticide resistance. However, the extent to which insecticide resistance impacts transmission is unclear. The objective of this study was to examine the association between the DDT/pyrethroid knockdown resistance mutation Vgsc-1014S, commonly termed kdr, and infection with Plasmodium falciparum sporozoites in Anopheles gambiae. METHODS: WHO standard methods were used to characterize susceptibility of wild female mosquitoes to 0.05 % deltamethrin. PCR-based molecular diagnostics were used to identify mosquitoes to species and to genotype at the Vgsc-L1014S locus. ELISAs were used to detect the presence of P. falciparum sporozoites and for blood meal identification. RESULTS: Anopheles mosquitoes were resistant to deltamethrin with mortality rates of 77.7 % [95 % CI 74.9-80.3 %]. Of 545 mosquitoes genotyped 96.5 % were A. gambiae s.s. and 3.5 % were Anopheles arabiensis. The Vgsc-1014S mutation was detected in both species. Both species were predominantly anthropophagic. In A. gambiae s.s., Vgsc-L1014S genotype was significantly associated with deltamethrin resistance (χ2 = 11.2; p < 0.001). The P. falciparum sporozoite infection rate was 4.2 %. There was a significant association between the presence of sporozoites and Vgsc-L1014S genotype in A. gambiae s.s. (χ2 = 4.94; p = 0.026). CONCLUSIONS: One marker, Vgsc-1014S, was associated with insecticide resistance and P. falciparum infection in wild-caught mixed aged populations of A. gambiae s.s. thereby showing how resistance may directly impact transmission.

Co-occurrence and distribution of East (L1014S) and West (L1014F) African knock-down resistance in Anopheles gambiae sensu lato population of Tanzania.

OBJECTIVE: Insecticide resistance molecular markers can provide sensitive indicators of resistance development in Anopheles vector populations. Assaying these makers is of paramount importance in the resistance monitoring programme. We investigated the presence and distribution of knock-down resistance (kdr) mutations in Anopheles gambiae s.l. in Tanzania. METHODS: Indoor-resting Anopheles mosquitoes were collected from 10 sites and tested for insecticide resistance using the standard WHO protocol. Polymerase chain reaction-based molecular diagnostics were used to genotype mosquitoes and detect kdr mutations. RESULTS: The An. gambiae tested were resistance to lambdacyhalothrin in Muheza, Arumeru and Muleba. Out of 350 An. gambiae s.l. genotyped, 35% were An. gambiae s.s. and 65% An. arabiensis. L1014S and L1014F mutations were detected in both An. gambiae s.s. and An. arabiensis. L1014S point mutation was found at the allelic frequency of 4-33%, while L1014F was at the allelic frequency 6-41%. The L1014S mutation was much associated with An. gambiae s.s. (χ(2) = 23.41; P < 0.0001) and L1014F associated with An. arabiensis (χ(2) = 11.21; P = 0.0008). The occurrence of the L1014S allele was significantly associated with lambdacyhalothrin resistance mosquitoes (Fisher exact P < 0.001). CONCLUSION: The observed co-occurrence of L1014S and L1014F mutations coupled with reports of insecticide resistance in the country suggest that pyrethroid resistance is becoming a widespread phenomenon among our malaria vector populations. The presence of L1014F mutation in this East African mosquito population indicates the spreading of this gene across Africa. The potential operational implications of these findings on malaria control need further exploration.

First molecular genotyping of voltage gated sodium channel alleles in Culex quinquefasciatus populations in Malaysia.

A nationwide investigation was performed to detect the presence of 1014 mutation(s) in voltage gated sodium channel (kdr) gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia. Molecular genotyping of kdr mutation was performed via a modified three tubes allele-specific-polymerase chain reaction (AS-PCR) and direct sequencing of kdr gene. Based on the results of AS-PCR, homozygous susceptible (SS) genotype was found in nine out of 14 populations with 38 individuals from a total sample size of 140. Heterozygous (RS) genotype was most predominant (99 individuals) and distributed across all study sites. Homozygous resistance (RR) genotype was detected in Perak (one individual) and Selangor (two individuals). The resistance kdr allele frequencies ranged from 0.1 to 0.55, with the highest being detected in Cx. quinquefasciatus population from Selangor. This study has documented the first field-evolved instance of 1014F mutation in Malaysian mosquitoes and the findings of this study could be utilized in the implementation of strategic measures in vector control programs in Malaysia.