Molecular analysis of knock down resistance (kdr) mutation and distribution of kdr genotypes in a wild population of Culex quinquefasciatus from India

Objective  To investigate the presence of knock down resistance ( kdr ) mutation, its frequency distribution in the principal vector of bancroftian filariasis, Culex quinquefasciatus from northeastern India, and to relate kdr genotypes with susceptibility and/or resistance to DDT and deltamethrin in this vectors.
Methods  Adult female mosquitoes were collected by aspiration from human dwellings in two villages, Benganajuli and Rikamari, and two military establishments, Field Units I and II. Insecticide susceptibility tests were performed following WHO methods with 4% DDT and 0.05% deltamethrin. Molecular identification of kdr mutation and genotyping of kdr locus was performed by allele-specific PCR (AS-PCR) and direct sequencing in a subset of samples.
Results  Mosquitoes were resistant to DDT and showed 11.9–41.2% mortality, whereas the knock down bioassay for deltamethrin suggests complete susceptibility to this insecticide in all study sites except Benganajuli. The result of AS-PCR confirmed the presence of three genotypes: susceptible (SS), resistant (RR) and heterozygous (SR) in the population. Genotype frequencies at kdr locus for DDT-resistant individuals conformed with the Hardy–Weinberg proportion, whereas DDT and deltamethrin susceptible individuals differed significantly ( P  < 0.05). The efficacy of AS-PCR in detecting the correct genotype was not encouraging.
Conclusions  This is the first report from India on kdr genotyping in C. quinquefasciatus , and it confirms the occurrence of kdr allele in this vector in northeastern India. This finding has serious implications for the filariasis control programmes in India.     

Absence of the kdr mutation in the molecular 'M' form suggests different pyrethroid resistance mechanisms in the malaria vector mosquito Anopheles gambiae s.s

Field tests conducted on adult Anopheles mosquitoes using standard WHO procedures, diagnostic kits and test papers in south-western Nigeria showed pyrethroid (deltamethrin and permethrin) resistance in adult populations of Anopheles gambiae sensu stricto. The knock-down resistance (kdr) mutation involved in pyrethroid resistance was only found in the molecular S form of An. gambiae s.s. even in area where both molecular M and S forms occurred in sympatry. The absence of the kdr mutation in the M form suggests an additional pyrethroid resistance mechanism in An. gambiae s.s.     

Knockdown resistance mutations (kdr) and insecticide susceptibility to DDT and pyrethroids in Anopheles gambiae from Equatorial Guinea

Objectives To determine the frequency of knockdown resistance (kdr) mutations in the malaria vector Anopheles gambiae s.s. from continental Equatorial Guinea; and to relate kdr genotypes with susceptibility to DDT and pyrethroid insecticides in this vector. Methods Female mosquitoes were collected in two villages, Miyobo and Ngonamanga, of mainland Equatorial Guinea. Insecticide susceptibility tests were performed following WHO procedures. Anopheles gambiae complex specimens were identified to species and molecular form by PCR. Genotyping of the kdr locus was performed by allele‐specific PCR and direct sequencing in a subset of samples. Results Both M and S molecular forms of A. gambiae were found in Ngonamanga whereas only the S‐form was identified in Miyobo. The two kdr mutations were detected in S‐form samples of both villages, with a higher frequency of the kdr‐e (Leu‐1014‐Ser) allele (Miyobo: 16%; Ngonamanga: 40%). The kdr‐w (Leu‐1014‐Phe) mutation was also detected in 3% of the M‐form. All individuals tested for pyrethroids were susceptible. A mortality rate of 86% was obtained for DDT. An overall kdr allele frequency (i.e. kdr‐e + kdr‐w) of 22% was detected in DDT resistant individuals, whereas susceptible individuals had a kdr frequency of 6%. Conclusion The co‐occurrence of both kdr mutations and reduced susceptibility to DDT found in A. gambiae highlights the importance of implementing efficient surveillance of insecticide resistance in Equatorial Guinea.     

安徽省中北部淡色库蚊对多种杀虫剂抗性及其kdr基因突变研究

目的 目的 了解安徽省中北部地区淡色库蚊对多种杀虫剂的抗药性现状及其kdr基因突变情况。方法 方法 2014年 6-9月在淮北市、 蚌埠市和滁州市3地采集淡色库蚊幼虫, 带回实验室饲养至成虫, 采用WHO成蚊接触筒药膜滤纸接触 法对其进行0.05%溴氰菊酯、 5%马拉硫磷、 0.1%噁虫威、 4%DDT抗性生物测定。以PCR扩增经溴氰菊酯抗性测定的库蚊 抗性相关kdr基因并进行测序, 统计L1014位点突变情况。结果 结果 上述3个地区淡色库蚊对溴氰菊酯、 马拉硫磷、 噁虫 威、 DDT 4种杀虫剂都产生了不同程度的抗药性, 对DDT产生的抗性较高; 虽然3地淡色库蚊接触DDT后的死亡率差异 无统计学意义 （F = 1.027,P > 0.05）; 但接触溴氰菊酯、 马拉硫磷、 噁虫威后的死亡率差异均有统计学意义 （F = 23.823、 33.955、 128.841, P均< 0.01）。3地淡色库蚊种群的kdr基因1014位点均存在L1014F、 L1014S 这2种非同义突变; L1014F 突变频率与溴氰菊酯抗性水平呈正相关 （r 2 = 0.718, P < 0.01）。结论 结论 安徽省中北部地区淡色库蚊对溴氰菊酯、 马拉硫 磷、 噁虫威、 DDT均产生了较强的抗性, kdr基因L1014F突变频率与溴氰菊酯抗性水平呈正相关; 各地区卫生部门需加强 对媒介蚊虫抗性的动态监测。     

First detection of a putative knockdown resistance gene in major mosquito vector, Aedes albopictus

The Asian tiger mosquito, Aedes albopictus (Skuse), is the major vector of Chikungunya fever and the secondary vector of dengue fever. We collected Ae. albopictus from Singapore and performed genotyping assay to detect mutations of the voltage-gated sodium channel, which is the target site of pyrethroid insecticides. We detected an amino acid substitution, F1534C, which is suspected to confer knockdown resistance (kdr) to pyrethroid insecticides. Of the collected mosquitoes, 53.8% were homozygous for this mutation, and the allele frequency of this mutation was estimated to be 73.1%. No kdr mutation was detected in the 5 other loci of domains II and IV. This is the first evidence for the presence of the kdr gene in Ae. albopictus, and our findings highlight the need for studying the global distribution of this allele in this important vector insect.     

Development and application of a simple colorimetric assay reveals widespread distribution of sodium channel mutations in Thai populations of Aedes aegypti

Dengue fever and its more serious complications dengue haemorrhagic fever and dengue shock syndrome are growing public health problems in tropical and subtropical countries. In the absence of a vaccine, most dengue control programmes rely heavily on the use of insecticides to target the Aedes mosquito vectors. As a limited number of insecticides are routinely used in control, monitoring for the presence of resistance is an essential component of dengue prevention programmes. The pyrethroid insecticides target the voltage-gated sodium channel on the insects’ neurons. Substitutions at residue 1016 of this protein have been associated with pyrethroid and DDT resistance in Aedes aegypti populations from Latin America and Asia. Here we report on the development of a simple colorimetric assay to detect these mutations in individual mosquitoes. Evaluation of this diagnostic assay on 180 Ae. aegypti individuals from Thailand revealed the presence of high frequencies of the Val1016Gly mutation throughout the country. The assay requires no specialised equipment and will enable monitoring for insecticide resistance associated alleles to be routinely incorporated into dengue surveillance operations.     

Distribution of the molecular forms of Anopheles gambiae and pyrethroid knock down resistance gene in Nigeria

We investigated the distribution of the molecular M and S forms of Anopheles gambiae and the knock down resistance (kdr) gene associated with pyrethroid and DDT resistance in A. gambiae s.s. at 13 localities across Nigeria. Two-three days old adult female mosquito reared from larval collections were tested using standard WHO procedures, diagnostic test kits and impregnated papers to assess their pyrethroid resistance status. Specimens were identified by PCR assays and characterized for the kdr gene. DNA from adult A. gambiae s.s. collected from human dwellings were also tested for the presence of the kdr gene. The overall collection was a mix of the molecular M and S forms across the mangrove (63:37%), forest (56:44%), and transitional (36:64%) ecotypes, but almost a pure collection of the S form in the Guinea and Sudan-savanna. Results of insecticide susceptibility tests showed that mosquitoes sampled at seven localities were susceptible to permethrin, deltamethrin, and DDT, but populations of A. gambiae resistant to these insecticides were recorded at six other localities mainly in the transitional and Guinea-savanna ecotypes. The kdr gene was found only in the molecular S forms, including areas where both forms were sympatric. The overall kdr frequency was low: <47% in forest, 37-48% in the transitional, and 45-53% in Guinea-savanna. The data suggest that pyrethroid resistance in A. gambiae in Nigeria is not as widespread when compared to neighbouring West African countries.     

安徽省沿淮地区中华按蚊溴氰菊酯抗性及代谢解毒酶活性kdr突变频率调查

目的 了解安徽省沿淮地区疟疾媒介中华按蚊对溴氰菊酯杀虫剂的抗性状况及谷胱甘肽S转移酶 （GSTs）、 细胞色素氧化酶 （P450s） 等代谢解毒酶活性和钠离子通道击倒抗性基因 （Knockdown resistance, kdr） 的突变情况。方法 2011 年8-9月采集安徽省蚌埠市李楼乡、 沫河口镇和沱湖乡中华按蚊成蚊样本, 采用WHO成蚊接触筒药膜滤纸接触法, 调查3 个地区中华按蚊对溴氰菊酯抗性状况。随机挑选样本采用生化法检测代谢解毒酶活性并用PCR扩增产物直接测序法检测分析钠离子通道kdr基因IIS6片段L1014位点基因型。结果 李楼乡、 沫河口镇和沱湖乡3个检测点的中华按蚊对溴氰菊酯60 min内击倒率分别为4.1%、 7.0%和8.2%, 恢复24 h后校正死亡率分别为8.2%、 12.0%、 12.8%, 均为抗性种群。3个检测点的中华按蚊GSTs和P450s活性均显著高于实验室敏感种群 （P < 0.001）。3个检测点的中华按蚊kdr基因均发生突变, 存在 L1014C和L1014F两种突变类型, 无野生纯合型 （TTG/TTG）, 实验室敏感种群kdr基因均为无突变的野生纯合型。结论 安徽省沿淮地区的中华按蚊已对溴氰菊酯产生较强的抗性, 代谢解毒酶活性比实验室敏感种群显著升高, 钠离子通道kdr基因L1014位点出现高频率的突变。     

中山市白纹伊蚊现场种群击倒抗性基因检测分析

目的 检测广东省中山市不同生境白纹伊蚊现场种群的击倒抗性(kdr)基因突变,了解其抗药性水平,探讨kdr基因突变与性别间抗药性差异的关系,为蚊虫的抗性机制研究以及科学防制提供依据.方法 2019年8-11月在中山市选择5个生境采集白纹伊蚊幼虫和蛹569只,实验室饲养至成虫,提取单只成蚊基因组DNA,扩增并测序分析kdr...     

Chlorfenapyr: a pyrrole insecticide for the control of pyrethroid or DDT resistant Anopheles gambiae (Diptera: Culicidae) mosquitoes

Owing to the development and spread of pyrethroid resistance in Anopheles gambiae in Africa there is an urgent need to develop alternative insecticides to supplement the pyrethroids. Chlorfenapyr is a pyrrole insecticide first commercialized for the control of agricultural pests and termites. Performance against An. gambiae bearing kdr (pyrethroid and DDT resistance) or Ace-1(R) insensitive acetylcholinesterase (organophosphate and carbamate resistance) mechanisms was studied using a variety of adult bioassay tests including a simulated-experimental hut system (tunnel tests) that allows uninhibited mosquito behaviour/insecticide interactions. Strains resistant to pyrethroids and organophosphates showed no cross resistance to chlorfenapyr. In cone bioassays on treated netting the mortality of adult mosquitoes showed an unexpected curvilinear response, with highest mortality occurring at intermediate dosages. Adults expressed irritability to chlorfenapyr at higher dosages, which might explain the dosage-mortality trend. Toxic activity of chlorfenapyr was slow compared to conventional neurotoxic insecticides and additional mortality occurred between 24h and 72 h. In tunnel tests, the dosage-mortality trend showed a more typical sigmoid response and most mortality occurred during the first 24h. Mosquito penetration through the holed, treated netting showed only limited inhibition and blood-feeding was not inhibited. Mortality rates in the kdr strain exposed to chlorfenapyr treated netting in tunnel tests were much higher than with permethrin treated netting over the same 100-500 mg/m(2) dosage range. Chlorfenapyr has potential for malaria control in treated-net or residual spraying applications in areas where mosquitoes are pyrethroid resistant. For treated-net applications chlorfenapyr might be combined with pyrethroid as a mixture to provide personal protection as well as to give control of resistant mosquitoes.     

Pyrethroid susceptibility in natural populations of the Anopheles punctulatus group (Diptera: Culicidae) in Papua New Guinea

The development of insecticide resistance has compromised mosquito control efforts in many parts of the world. Papua New Guinea (PNG) has a long history of dichlorodiphenyltrichloroethane (DDT) use and currently distributes pyrethroid-treated nets for malaria control. This study is the first to investigate the status of pyrethroid resistance in the Anopheles punctulatus group, the major malaria and filariasis vectors of PNG. The study used World Health Organization standard susceptibility bioassays to detect knockdown phenotypes and a novel nested polymerase chain reaction to detect the knockdown resistant (kdr) allele in these vectors. Our results show 100% susceptibility to pyrethroids in all populations surveyed and an absence of the kdr allele.     

云南省埃及伊蚊对拟除虫菊酯类抗性群体的击倒抗性基因突变分析

目的 检测云南省登革热重点地区对氯菊酯和高效氯氟氰菊酯杀虫剂已产生抗性的埃及伊蚊的击倒抗性(knockdown resistance,kdr)基因,进行基因突变分析,阐明抗性表型与kdr基因突变的关系。方法 收集云南省景洪市、勐腊县、勐海县、瑞丽市和耿马县经过氯菊酯和高效氯氟氰菊酯杀虫剂测定的埃及伊蚊成蚊样本,分别提取单蚊基因组DNA,Allele-specific PCR(AS-PCR)扩增和分析其kdr部分基因片段,统计抗性与敏感表型中kdr突变的基因型和基因频率。结果 共580份雌性埃及伊蚊样本被用于V1016G和F1534C击倒抗性突变的检测。V1016G的突变频率为99.83%(579/580),F1534C的突变频率为46.38%(269/580)。V1016G和F1534C突变频率差异存在统计学意义(χ²=421.338,P=0.000)。5个埃及伊蚊种群中,V1016G突变率除了瑞丽市外,其他区域均达到100.00%。F1534C突变率景洪市最高为84.16%,耿马县最低为5.66%。抗性表型与敏感表型的V1016G和F1534C突变率差异均存在统计学意义(χ²=7.298,P=0.007;χ²=14.010,P=0.000)。580份样本中,有65份样本同时存在V1016G和F1534C位点突变,突变率为11.21%;不同地区同时突变率范围为0~33.66%,景洪市同时突变率最高为33.66%。结论 云南省登革热重点地区埃及伊蚊种群存在V1016G和F1534C突变,V1016G突变率高且分布广,F1534C突变以景洪市埃及伊蚊种群为最高。敏感表型与抗性表型V1016G和F1534C突变存在差异,提示抗性产生与突变存在一定的关系。     

Frequency of Val1016Ile mutation in the voltage-gated sodium channel gene of Aedes aegypti Brazilian populations

One of the major insect pyrethroid resistance mechanisms affects its target site, the voltage-gated sodium channel (Na_v). In Aedes aegypti , the Val1016Ile substitution of the AaNa _v gene is associated to resistance in several Latin American countries. Genotyping of susceptible and resistant mosquitoes from seven Brazilian localities detected the 1016Ile mutation in five populations with a higher frequency of this substitution in resistant specimens in all cases. Furthermore, analysis of nine additional field populations revealed that five also presented the 1016Ile mutation. Our data suggest a recent dissemination and involvement of this substitution with pyrethroid resistance in Brazil.     

乙肝病毒C基因启动子变异与乙肝病情的关系

研究乙型肝炎病毒（HBV)C基因启动子（CP）变异与无症状慢性HBV携带者（AsC)肝炎发作及与慢性乙肝病情的关系。．通过PCR及其产物直接测序，检测4例AsC、27例慢性乙肝和3例慢性重型乙肝患者血清的HBV CP序列，并定量测定血清的HBV DNA。（1）CP主要变异为nt1726-1730聚集（1726A→C、1727A →T、1730C→G）和nt1762 1764双变异（1762A→T和1764G→A）。（2）CP聚集变与AsC首次肝炎 急性发作有关。11例中，8例出现CP聚集变异。且1例在AsC状态时无CP变异，肝炎发作时出现CP聚集变异。（3）CP聚集变异合并CP双突变的乙肝患者,表现为重型肝炎或迅速进展为肝硬化，HBV DNA高水平；HBeAg／抗HBe转移。CP聚集变异与AsC肝炎急性发作有关；CP聚集变异 与CP双变异同时存在，使慢性乙肝病情加重。     

Dynamics of the pyrethroid knockdown resistance allele in western Kenyan populations of Anopheles gambiae in response to insecticide-treated bed net trials

Permethrin and DDT resistance in Anopheles gambiae s.s. associated with a leucine-serine knockdown resistance (kdr) mutation in the voltage-gated sodium channel gene was discovered recently in western Kenya where a large scale permethrin-impregnated bed net (ITN) program has been implemented. Collections of An. gambiae s.l. were made from intervention and control villages prior to and after onset of the program. The kdr genotypes were determined using allele-specific polymerase chain reaction diagnostic tests. In An. gambiae s.s., the frequency of the kdr mutation prior to ITN introduction was approximately 3-4% in western Kenya and zero in samples from the coast. After ITN introduction, the kdr mutation increased in ITN and neighboring villages from approximately 4% to approximately 8%, but remained unchanged in villages at least 20 km distant and was not detected in coastal Kenya. The identical leucine-serine mutation was found in a single An. arabiensis individual among 658 tested. The leucine-phenylalanine kdr mutation common in west African An. gambiae populations was not detected in An. gambiae s.l. from Kenya. Implications for the population structure and control of An. gambiae are discussed.     

A new high-performance PCR diagnostic for the detection of pyrethroid knockdown resistance kdr in Anopheles gambiae

Monitoring the spread of the knockdown resistance allele kdr in areas of extensive pyrethroid use is critical to vector-control projects. Currently available methods for detecting kdr from DNA samples are characterized by poor amplification, time-consuming steps, and primers that exhibit frequent null alleles. We describe a new PCR diagnostic that uses fluorescent primers based on conserved priming sites and enables simple detection of the kdr allele on a sequencer. Using samples from a West African Anopheles gambiae population, we show that the new PCR yielded significantly higher rates of amplification and more accurate estimates of kdr frequency. The method works equally well for the leucine to phenylalanine substitution found in West Africa and the East African leucine to serine substitution.     

A multi-center study in order to further define the molecular basis of beta-thalassemia in Thailand, Pakistan, Sri Lanka, Mauritius, Syria, and India, and to develop a simple molecular diagnostic strategy by amplification refractory mutation system-polymerase chain reaction.

The spectrum of the beta-thalassemia mutations of Thailand, Pakistan, India, Sri Lanka, Mauritius and Syria has been further characterized by a multi-center study of 1,235 transfusion-dependent patients, and the mutations discovered used to assess the fidelity of a simple diagnostic strategy. A total of 44 beta-thalassemia mutations were identified either by allele-specific oligonucleotide hybridization, amplification with allele-specific primers, or DNA sequencing of amplified product. The results confirm and extend earlier findings for Thailand, Pakistan, India, Mauritius and Syria. This is the first detailed report of the spectrum of mutations for Sri Lanka. Two novel mutations were identified, codon 55 (-A) and IVS-I-129 (A-->C), both found in Sri Lankan patients. Two beta-thalassemia mutations were found to coexist in one beta-globin gene: Sri Lankan patients homozygous for the beta0 codon 16 (-C) frameshift were also homozygous for the beta+ codon 10 (C-->A) mutation. Studies of Sri Lankan, Pakistani, and Indian carriers suggest the codon 10 (C-->A) mutation is just a rare polymorphism on an ancestral allele, on which the beta0 codon 16 (-C) mutation has arisen. Each country was found to have only a few common mutations accounting for 70% or more of the beta-thalassemia alleles. A panel of primers to diagnose the majority of the mutations by the amplification refractory mutation system was developed, enabling a simple molecular diagnostic strategy to be introduced for each country participating in the multi-center study.     

Longitudinal survey of knockdown resistance to pyrethroid (kdr) in Mali, West Africa, and evidence of its emergence in the Bamako form of Anopheles gambiae s.s

Studies aimed at monitoring the spread of knockdown resistance to pyrethroids (kdr) in time and space are particularly useful for detecting barriers to gene flow among the chromosomal and molecular forms of Anopheles gambiae. We used a recently developed polymerase chain reaction assay to estimate changes in kdr frequency that occurred in several mixed-form populations from Mali, West Africa, in the past decade. We found that the kdr allele significantly increased in frequency in most populations but was still absent from the M molecular form. Importantly, within the S molecular form, kdr was detected for the first time in the Bamako chromosomal form. These results provide important insights on the patterns of spread and emergence of pyrethroid knockdown resistance in West Africa.     

河南省中华按蚊抗药性及其与kdr突变关联性的初步研究

目的 了解河南省主要传疟媒介中华按蚊对拟除虫菊酯类杀虫剂的抗药性及其与击倒抗性基因型的关联性.方法 2009年以区分剂量法调查河南省桐柏、淮滨和永城3地中华按蚊对溴氰菊酯和氟氯氰菊酯的抗药性,并抽取部分样本进行了kdr基因序列检测.χ2检验判断其抗药性与kdr基因突变的关联性.结果 河南省3县(市)中华按蚊溴氰菊酯和氟...     

A MULTI-CENTER STUDY IN ORDER TO FURTHER DEFINE THE MOLECULAR BASIS OF β-THALASSEMIA IN THAILAND,PAKISTAN, SRI LANKA,MAURITIUS, SYRIA,AND INDIA,AND TO DEVELOP A SIMPLE MOLECULAR DIAGNOSTIC STRATEGY BY AMPLIFICATION REFRACTORY MUTATION SYSTEM-POLYMERASE CHAIN REACTION

The spectrum of the β-thalassemia mutations of Thailand, Pakistan, India, Sri Lanka, Mauritius and Syria has been further characterized by a multi-center study of 1,235 transfusion-dependent patients, and the mutations discovered used to assess the fidelity of a simple diagnostic strategy. A total of 44 β-thalassemia mutations were identified either by allele-specific oligonucleotide hybridization, amplification with allele-specific primers, or DNA sequencing of amplified product. The results confirm and extend earlier findings for Thailand, Pakistan, India, Mauritius and Syria. This is the first detailed report of the spectrum of mutations for Sri Lanka. Two novel mutations were identified, codon 55 (?A) and IVS-I-129 (A → C), both found in Sri Lankan patients. Two β-thalassemia mutations were found to coexist in one β-globin gene: Sri Lankan patients homozygous for the β⁰ codon 16 (?C) frameshift were also homozygous for the β⁺ codon 10 (C → A) mutation. Studies of Sri Lankan, Pakistani, and Indian carriers suggest the codon 10 (C → A) mutation is just a rare polymorphism on an ancestral allele, on which the β⁰ codon 16 (?C) mutation has arisen. Each country was found to have only a few common mutations accounting for 70% or more of the β-thalassemia alleles. A panel of primers to diagnose the majority of the mutations by the amplification refractory mutation system was developed, enabling a simple molecular diagnostic strategy to be introduced for each country participating in the multi-center study.