Development of Deltamethrin-Laced Attractive Toxic Sugar Bait to Control Aedes aegypti (Linnaeus) Population.

Background: The attractive toxic sugar bait (ATSB) is a promising strategy for controlling mosquitoes at the adult stage. The strategy is based on the use of a combination of fruit juice, sugar, and a toxin in order to attract and kill the adult mosquitoes. The selection of the components and optimization of their concentrations is significant for the formulation of an effective ATSB. Methods: The present study formulated nine ATSBs and evaluated their efficacy against two laboratory strains (AND-Aedes aegypti and AND-Aedes aegypti-DL10) and two wildcaught colonized strains of Aedes aegypti (GVD-Delhi and SHD-Delhi). Initially, nine attractive sugar baits (ASBs) were prepared using a mixture of 100% fermented guava juice (attractant) with 10% sucrose solution (w/v) in 1 : 1 ratio. ATSBs were formulated by mixing each ASB with different concentrations of deltamethrin in the ratio of 9 : 1 to obtain final deltamethrin concentration of 0.003125-0.8 mg/10 mL ATSB. Cage bioassays were conducted with 50 mosquitoes for 24 h in order to evaluate the efficacy of each ATSB against the four strains of Ae. aegypti. The data were statistically analyzed using PASW software 19.0 program and 2-way ANOVA. Results: The ATSB formulations registered 8.33-97.44% mortality against AND-Aedes aegypti and 5.15-96.91% mortality against AND-Aedes aegypti-DL10 strains of Ae. aegypti, while GVD-Delhi strain registered 2.04-95.83% mortality and SHD-Delhi strain showed 5.10-97.96% mortality. The administration of 0.8 mg of deltamethrin within 10 mL of attractive toxic sugar bait (ATSB) has led to the maximum mortality rate in adult mosquitoes. Conclusions: The ATSBs formulated with guava juice-ASB and deltamethrin (9 : 1) showed toxin dose-dependent toxicity by all the four strains of Ae. aegypti. Most effective dosage was found as 0.8 mg deltamethrin/10 mL ATSB which imparted 96% to 98% mortality in adult mosquitoes. The investigations demonstrated the efficacy of deltamethrin-laced ATSB formulations against Ae. aegypti and highlighted the need for conduct of structured field trials and investigating the impact on disease vectors and nontarget organisms.

First Report of Anopheles annularis s.l., An. maculatus s.s., and An. culicifacies s.l. as Malaria Vectors and a New Occurrence Record for An. pseudowillmori and An. sawadwongporni in Alipurduar District Villages, West Bengal, India.

A comprehensive entomological survey was undertaken in Alipurduar District, West Bengal, from 2018 to 2020 and in 2022. This study was prompted by reported malaria cases and conducted across nine villages, seven Sub-Centres, and three Primary Health Centres (PHCs). Mosquitoes were hand-collected with aspirators and flashlights from human dwellings and cattle sheds during the daytime. Both morphological and molecular techniques were used for species identification. Additionally, mosquitoes were tested for Plasmodium parasites and human blood presence. Mosquito species such as An. barbirostris s.l., An. hyrcanus s.l., An. splendidus, and An. vagus were morphologically identified. For species like An. annularis s.l., An. minimus s.s., An. culicifacies s.l., and An. maculatus s.s., a combination of morphological and molecular techniques was essential. The mitochondrial cytochrome c oxidase gene subunit 1 (CO1) was sequenced for An. annularis s.l., An. maculatus s.s., An. culicifacies s.l., An. vagus, and some damaged samples, revealing the presence of An. pseudowillmori and An. fluviatilis. The major Anopheles species were An. annularis s.l., An. culicifacies s.l., and An. maculatus s.s., especially in Kumargram and Turturi PHCs. Plasmodium positivity was notably high in An. annularis s.l. and An. maculatus s.s. with significant human blood meal positivity across most species. Morphological, molecular, and phylogenetic analyses are crucial, especially for archived samples, to accurately identify the mosquito fauna of a region. Notably, this study confirms the first occurrence of An. pseudowillmori and An. sawadwongporni in West Bengal and implicates An. maculatus s.s., An. culicifacies s.l., and An. annularis s.l. as significant vectors in the Alipurduar region.

Laboratory evaluation of the efficacy of deltamethrin-laced attractive toxic sugar bait formulation on Anopheles stephensi.

BACKGROUND: Attractive toxic sugar bait (ATSB) is a promising "attract and kill"-based approach for mosquito control. It is a combination of flower nectar/fruit juice to attract the mosquitoes, sugar solution to stimulate feeding, and a toxin to kill them. Selecting an effective attractant and optimizing concentration of toxicant is significant in the formulation of ATSB. METHODS: Current study formulated an ATSB using fruit juice, sugar and deltamethrin, a synthetic pyrethroid. It was evaluated against two laboratory strains of Anopheles stephensi. Initial studies evaluated comparative attractiveness of nine different fruit juices to An. stephensi adults. Nine ASBs were prepared by adding fermented juices of plum, guava, sweet lemon, orange, mango, pineapple, muskmelon, papaya, and watermelon with 10% sucrose solution (w/v) in 1:1 ratio. Cage bioassays were conducted to assess relative attraction potential of ASBs based on the number of mosquito landings on each and the most effective ASB was identified. Ten ATSBs were prepared by adding the identified ASB with different deltamethrin concentrations (0.015625-8.0 mg/10 mL) in 1:9 ratio. Each ATSB was assessed for the toxic potential against both the strains of An. stephensi. The data was statistically analysed using PASW (SPSS) software 19.0 program. RESULTS: The cage bioassays with nine ASBs revealed higher efficacy (p < 0.05) of Guava juice-ASB > Plum juice-ASB > Mango juice-ASB in comparison to rest of the six ASB's. The bioassay with these three ASB's ascertained the highest attractancy potential of guava juice-ASB against both the strains of An. stephensi. The ATSB formulations resulted in 5.1-97.9% mortality in Sonepat (NIMR strain) with calculated LC30, LC50, and LC90 values of 0.17 mg deltamethrin/10 mL, 0.61 mg deltamethrin/10 mL, and 13.84 mg deltamethrin/10 mL ATSB, respectively. Whereas, 6.12-86.12% mortality was recorded in the GVD-Delhi (AND strain) with calculated LC30, LC50, and LC90 values of 0.25 mg deltamethrin/10 mL, 0.73 mg deltamethrin/10 mL and 10.22 mg deltamethrin/10 mL ATSB, respectively. CONCLUSION: The ATSB formulated with guava juice-ASB and deltamethrin (0.0015625-0.8%) in 9:1 ratio showed promising results against two laboratory strains of An. stephensi. Field assessment of these formulations is being conducted to estimate their feasibility for use in mosquito control.

A new WHO bottle bioassay method to assess the susceptibility of mosquito vectors to public health insecticides: results from a WHO-coordinated multi-centre study.

BACKGROUND: The continued spread of insecticide resistance in mosquito vectors of malaria and arboviral diseases may lead to operational failure of insecticide-based interventions if resistance is not monitored and managed efficiently. This study aimed to develop and validate a new WHO glass bottle bioassay method as an alternative to the WHO standard insecticide tube test to monitor mosquito susceptibility to new public health insecticides with particular modes of action, physical properties or both. METHODS: A multi-centre study involving 21 laboratories worldwide generated data on the susceptibility of seven mosquito species (Aedes aegypti, Aedes albopictus, Anopheles gambiae sensu stricto [An. gambiae s.s.], Anopheles funestus, Anopheles stephensi, Anopheles minimus and Anopheles albimanus) to seven public health insecticides in five classes, including pyrethroids (metofluthrin, prallethrin and transfluthrin), neonicotinoids (clothianidin), pyrroles (chlorfenapyr), juvenile hormone mimics (pyriproxyfen) and butenolides (flupyradifurone), in glass bottle assays. The data were analysed using a Bayesian binomial model to determine the concentration-response curves for each insecticide-species combination and to assess the within-bioassay variability in the susceptibility endpoints, namely the concentration that kills 50% and 99% of the test population (LC50 and LC99, respectively) and the concentration that inhibits oviposition of the test population by 50% and 99% (OI50 and OI99), to measure mortality and the sterilizing effect, respectively. RESULTS: Overall, about 200,000 mosquitoes were tested with the new bottle bioassay, and LC50/LC99 or OI50/OI99 values were determined for all insecticides. Variation was seen between laboratories in estimates for some mosquito species-insecticide combinations, while other test results were consistent. The variation was generally greater with transfluthrin and flupyradifurone than with the other compounds tested, especially against Anopheles species. Overall, the mean within-bioassay variability in mortality and oviposition inhibition were < 10% for most mosquito species-insecticide combinations. CONCLUSION: Our findings, based on the largest susceptibility dataset ever produced on mosquitoes, showed that the new WHO bottle bioassay is adequate for evaluating mosquito susceptibility to new and promising public health insecticides currently deployed for vector control. The datasets presented in this study have been used recently by the WHO to establish 17 new insecticide discriminating concentrations (DCs) for either Aedes spp. or Anopheles spp. The bottle bioassay and DCs can now be widely used to monitor baseline insecticide susceptibility of wild populations of vectors of malaria and Aedes-borne diseases worldwide.

Multi-country evaluation of the durability of pyrethroid plus piperonyl-butoxide insecticide-treated nets: study protocol.

BACKGROUND: Mass distributions of long-lasting insecticidal nets (LLINs) have contributed to large reductions in the malaria burden. However, this success is in jeopardy due in part to the increasing pyrethroid-resistant mosquito population as well as low LLINs coverage in various areas because the lifespan of LLINs is often shorter than the interval between replenishment campaigns. New insecticide-treated nets (ITNs) containing pyrethroid and piperonyl-butoxide (PBO) have shown a greater reduction in the incidence of malaria than pyrethroid LLINs in areas with pyrethroid-resistant mosquitoes. However, the durability (attrition, bio-efficacy, physical integrity and chemical retainment) of pyrethroid-PBO ITNs under operational settings has not been fully characterized. This study will measure the durability of pyrethroid-PBO ITNs to assess whether they meet the World Health Organization (WHO) three years of operational performance criteria required to be categorized as "long-lasting". METHODS: A prospective household randomized controlled trial will be conducted simultaneously in Tanzania, India and Côte d'Ivoire to estimate the field durability of three pyrethroid-PBO ITNs (Veeralin®, Tsara® Boost, and Olyset® Plus) compared to a pyrethroid LLIN: MAGNet®. Durability monitoring will be conducted up to 36 months post-distribution and median survival in months will be calculated. The proportion of ITNs: (1) lost (attrition), (2) physical integrity, (3) resistance to damage score, (4) meeting WHO bio-efficacy (≥ 95% knockdown after 1 h or ≥ 80% mortality after 24 h for WHO cone bioassay, or ≥ 90% blood-feeding inhibition or ≥ 80% mortality after 24 h for WHO Tunnel tests) criteria against laboratory-reared resistant and susceptible mosquitoes, and insecticidal persistence over time will be estimated. The non-inferiority of Veeralin® and Tsara® Boost to the first-in-class, Olyset® Plus will additionally be assessed for mortality, and the equivalence of 20 times washed ITNs compared to field aged ITNs will be assessed for mortality and blood-feeding inhibition endpoints in the Ifakara Ambient Chamber Test, Tanzania. CONCLUSION: This will be the first large-scale prospective household randomized controlled trial of pyrethroid-PBO ITNs in three different countries in East Africa, West Africa and South Asia, simultaneously. The study will generate information on the replenishment intervals for PBO nets.

Insecticide resistance status of malaria vectors in the malaria endemic states of India: implications and way forward for malaria elimination.

Background: In 2012, the World Health Organization (WHO) released the Global Plan for Insecticide Resistance Management in malaria vectors to stress the need to address insecticide resistance. In a prospective multi-centric study commissioned by the Indian Council of Medical Research (ICMR), we assessed the insecticide susceptibility status of the primary malaria vectors in India from 2017 through 2019. Methods: The insecticide susceptibility status of the prevalent primary malaria vectors - An. culicifacies, An. fluviatilis, An. stephensi, An. minimus and An. baimaii and secondary malaria vectors - An. aconitus, An. annularis and An. philippinensis/nivepes from 328 villages in 79 districts of 15 states of India were assessed following the WHO method mainly to insecticides used in vector control, organochlorine (DDT), organophosphate (malathion), and other pyrethroids (alpha-cypermethrin, cyfluthrin, lambda-cyhalothrin and permethrin). The study sites were selected as suggested by the National Vector Borne Disease Control Programme. Results: The primary malaria vector An. culicifacies showed resistance to DDT (50/50 districts including two districts of Northeastern India), malathion (27/44 districts), and deltamethrin (17/44 districts). This species was resistant to DDT alone in 19 districts, double resistant to DDT-malathion in 16 districts, double resistant to DDT-deltamethrin in 6 districts, and triple resistant to DDT-malathion-deltamethrin in 9 districts. An. minimus and An. baimaii were susceptible in Northeastern India while An. fluviatilis and the secondary malaria vector An. annularis was resistant to DDT in Jharkhand. Conclusion: In this study we report that among the primary vectors An. culicifacies is predominantly resistant to multiple insecticides. Our data suggest that periodic monitoring of insecticide susceptibility is vital. The national malaria program can take proactive steps for insecticide resistance management to continue its push toward malaria elimination in India.

First Report on Intensity Bioassays for Pyrethroid Resistance in Anopheles culicifacies s.l in District Dindori of Madhya Pradesh State and Districts Kanker and Bastar of Chhattisgarh State, India.

Background: The major malaria vector in India is Anopheles culicifacies, and indoor residual spraying (IRS) and distribution of long-lasting insecticidal nets (LLINs) are the two main vector control measures in the national program. This species has shown resistance to dichlorodiphenyltrichloroethane (DDT), malathion, and synthetic pyrethroids (SP). The study was carried out in three districts, that is, Dindori of Madhya Pradesh and Kanker and Bastar from Chhattisgarh state to know the range of resistance phenotypes and to assess the strength of resistance in An. culicifacies. Methods: An. culicifacies collected from the field was tested to determine the susceptibility status to the discriminating concentration (DC) of different insecticides, that is, DDT 4.0%, malathion 5.0%, alphacypermethrin 0.05%, and deltamethrin 0.05% following the World Health Organization (WHO) procedures. Further, intensity bioassays of the resistant An. culicifacies to 1X discriminating concentration (DC) of alphacypermethrin and deltamethrin were conducted by exposing 5X and 10X concentrations of 1X DC. Results are interpreted as per the WHO criterion. Results: The overall result of susceptibility status in An. culicifacies in districts Dindori, Kanker, and Bastar revealed confirmed resistance to DDT, malathion, alphacypermethrin, and deltamethrin registering mortality of 15% (10-20%), 70% (65-75%), 78.6% (77-82.5%), and 87% (84.8-91.3%), respectively. Further, in district Dindori and Baster, the intensity bioassay test at 5X DC of alphacypermethrin and deltamethrin revealed 99% and 100%, respectively, while in district Kanker, the species registered moderate resistance with 92.5% and 95% mortality, respectively, in 5X DC of alphacypermethrin and deltamethrin. However, in 10X DC, the An. culicifacies was susceptible to both pyrethroids (100%) in district Kanker. Conclusion: The results of the intensity bioassay tests with SP-resistant An. culicifacies suggested no change of insecticide is required in the ongoing intervention. However, regular monitoring of insecticide susceptibility and intensity bioassays in malaria vectors in view of continued use of these interventions may increase resistance and for implementing effective vector management strategies.

Attractive Sugar Bait Formulation for Development of Attractive Toxic Sugar Bait for Control of Aedes aegypti (Linnaeus).

Background: Attractive toxic sugar bait (ATSB), based on "attract and kill" approach, is a novel and promising strategy for mosquito control. Formulation of an attractive sugar bait (ASB) solution by selecting an efficient olfaction stimulant and preparation of an optimized sugar-attractant dosage is a significant component for the success of the approach. Methods: Current study evaluated relative potential of nine ASBs, formulated by combination of sugar and fresh fruit juices (guava, mango, muskmelon, orange, papaya, pineapple, plum, sweet lemon, and watermelon) in attracting Aedes aegypti adults. Freshly extracted and 48-hour-fermented juices were combined with 10% sucrose solution (w/v) in 1 : 1 ratio. Cage bioassays were conducted against two laboratory strains (susceptible: AND-Aedes aegypti; deltamethrin-selected: AND-Aedes aegypti-DL10) and two field-collected strains (Shahdara strain of Aedes aegypti: SHD-Delhi; Govindpuri strain of Aedes aegypti: GVD-Delhi). Each of the nine ASBs was assayed, individually or in groups of three, for its attraction potential based on the relative number of mosquito landings. The data were analysed for statistical significance using PASW (SPSS) software 19.0 program. Results: The prescreening bioassay with individual ASB revealed significantly higher efficacy of ASB containing guava/plum/mango juice than that containing six other juices (p < 0.05) against both the laboratory and field strains. The bioassay with three ASBs kept in one cage, one of the effective ASBs and two others randomly selected ASBs, also showed good attractancy of the guava/plum/mango juice-ASB (p < 0.05). The postscreening assays with these three ASBs revealed maximum attractant potential of guava juice-sucrose combination for all the four strains of Ae. aegypti. Conclusion. Guava juice-ASB showed the highest attractancy against both laboratory and field-collected strains of Ae. aegypti and can be used to formulate ATSB by combining with a toxicant. The field studies with these formulations will ascertain their efficacy and possible use in mosquito management programs.

Monitoring of Insecticide Resistance in Anopheles culicifacies in Twelve Districts of Madhya Pradesh, Central India (2017-2019).

BACKGROUND: Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are malaria vector control measures used in India, but the development of insecticide resistance poses major impediments for effective vector control strategies. As per the guidelines of the National Vector Borne Disease Control Programme (NVBDCP), the study was conducted in 12 districts of Madhya Pradesh to generate data on insecticide resistance in malaria vectors. METHODS: The susceptibility tests were conducted on adult An. culicifacies as per the WHO standard technique with wild-caught mosquitoes. The blood-fed female mosquitoes were exposed in 3 to 4 replicates on each occasion to the impregnated papers with specified discriminating dosages of the insecticides (DDT: 4%, malathion: 5%, deltamethrin: 0.05%, and alphacypermethrin: 0.05%), for one hour, and mortality was recorded after 24-hour holding. RESULTS: An. culicifacies was found resistant to DDT 4% in all the 12 districts and malathion in 11 districts. The resistance to alphacypermethrin was also observed in two districts, and possible resistance was found to alphacypermethrin in seven districts and to deltamethrin in eight districts, while the vector was found susceptible to both deltamethrin and alphacypermethrin in only 3 districts. CONCLUSION: An. culicifacies is resistant to DDT and malathion and has emerging resistance to pyrethroids, alphacypermethrin, and deltamethrin. Therefore, regular monitoring of insecticide susceptibility in malaria vectors is needed for implementing effective vector management strategies. However, studies to verify the impact of IRS with good coverage on the transmission of disease are required before deciding on the change of insecticide in conjunction with epidemiological data.

Bionomics of Anopheles culicifacies Sensu Lato in two Malaria Endemic Districts of Central Gujarat, India.

Background: Gujarat State has been witnessing large scale urbanization, in last two decades, resulting changes in local environment and microclimate may have also influenced the resting, feeding habits and development of Anopheles culicifacies sensu 1ato. Therefore, a systematic longitudinal study was undertaken to know the bionomics of An. culicifacies s.l. in present study. Methods: The study was conducted in four sentinel villages in Kheda and Panchmahal Districts. The mosquitoes resting indoors and outdoors were collected in early morning hours, using mouth aspirator, pyrethrum space spray and light traps. Mosquito landing collections on human volunteers was carried out from dusk to dawn. Species composition, abundance, seasonal prevalence, resting behavior (Endophily and Exophily), sibling species composition, vector potential and insecticide susceptibility status of malaria vectors was studied. Results: Six Anopheles species were collected, An. subpictus s.l. was the predominant species followed by An. culicifacies s.l., a known malaria vector was resting indoor and zoophagic behaviour. Anopheles culicifacies, sibling species B (89%) was found. The sporozoite rate (%) and entomological inoculation rate in Kheda was 2.33%, 3.09 per bite/person/annum and they were 1.05% and 0.475 bite/person/annum in Panchmahal, respectively. Anopheles culicifacies s.l. was found possible resistance to alpha-cypermethrin. Conclusion: Anopheles culicifacies s.l. showed endophillic, zoophagic behaviour and found possible resistance to alpha-cypermethrin. Early biting behaviour of An. culicifacies s.l. in this area is a cause of concern. Therefore, there is need for frequent monitoring and evaluation of vector control measures in order to achieve the elimination target of malaria in this area.

Correction: Contemporary status of insecticide resistance in the major Aedes vectors of arboviruses infecting humans.

[This corrects the article DOI: 10.1371/journal.pntd.0005625.].

Biology and bionomics of malaria vectors in India: existing information and what more needs to be known for strategizing elimination of malaria.

India has committed to eliminate malaria by 2030. The national framework for malaria elimination released by the Government of India plans to achieve this goal through strategic planning in a phased manner. Since vector control is a major component of disease management and vector elimination, it requires a thorough understanding of the biology and bionomics of malaria vectors exhibiting definite distribution patterns in diverse ecosystems in the country. Although a wealth of information is available on these aspects, lesser-known data are on biting time and rhythm, and the magnitude of outdoor transmission by the vectors which are crucial for effective implementation of the key vector control interventions. Most of the data available for the vector species are at sensu lato level, while the major vectors are species complexes and their members distinctly differ in biological characters. Furthermore, the persistent use of insecticides in indoor residual spray and long-lasting insecticidal nets has resulted in widespread resistance in vectors and changes in their behaviour. In this document, challenges in vector control in the Indian context have been identified and possible solutions to overcome the problem are suggested. Adequate addressing of the issues raised would greatly help make a deep dent in malaria transmission and consequently result in disease elimination within the targeted time frame.

Management of insecticide resistance in the major Aedes vectors of arboviruses: Advances and challenges.

BACKGROUND: The landscape of mosquito-borne disease risk has changed dramatically in recent decades, due to the emergence and reemergence of urban transmission cycles driven by invasive Aedes aegypti and Ae. albopictus. Insecticide resistance is already widespread in the yellow fever mosquito, Ae. Aegypti; is emerging in the Asian tiger mosquito Ae. Albopictus; and is now threatening the global fight against human arboviral diseases such as dengue, yellow fever, chikungunya, and Zika. Because the panel of insecticides available for public health is limited, it is of primary importance to preserve the efficacy of existing and upcoming active ingredients. Timely implementation of insecticide resistance management (IRM) is crucial to maintain the arsenal of effective public health insecticides and sustain arbovirus vector control. METHODOLOGY AND PRINCIPAL FINDINGS: This Review is one of a series being generated by the Worldwide Insecticide resistance Network (WIN) and aims at defining the principles and concepts underlying IRM, identifying the main factors affecting the evolution of resistance, and evaluating the value of existing tools for resistance monitoring. Based on the lessons taken from resistance strategies used for other vector species and agricultural pests, we propose a framework for the implementation of IRM strategies for Aedes mosquito vectors. CONCLUSIONS AND SIGNIFICANCE: Although IRM should be a fixture of all vector control programs, it is currently often absent from the strategic plans to control mosquito-borne diseases, especially arboviruses. Experiences from other public health disease vectors and agricultural pests underscore the need for urgent action in implementing IRM for invasive Aedes mosquitoes. Based on a plan developed for malaria vectors, here we propose some key activities to establish a global plan for IRM in Aedes spp.

Second WIN International Conference on "Integrated approaches and innovative tools for combating insecticide resistance in vectors of arboviruses", October 2018, Singapore.

The past 40 years have seen a dramatic emergence of epidemic arboviral diseases transmitted primarily by mosquitoes. The frequency and magnitude of the epidemics, especially those transmitted by urban Aedes species, have progressively increased over time, accelerating in the past 10 years. To reduce the burden and threat of vector-borne diseases, the World Health Organization (WHO) has recently adopted the Global Vector Control Response (GVCR) in order to support countries in implementing effective sustainable vector control. The evidence-base to support vector control is however limited for arboviral diseases which make prioritization difficult. Knowledge gaps in the distribution, mechanisms and impact of insecticide resistance on vector control impedes the implementation of locally tailored Aedes control measures. This report summarizes the main outputs of the second international conference of the Worldwide Insecticide resistance Network (WIN) on "Integrated approaches and innovative tools for combating insecticide resistance in arbovirus vectors" held in Singapore, 1-3 October 2018. The aims of the conference were to review progress and achievements made in insecticide resistance surveillance worldwide, and to discuss the potential of integrated vector management and innovative technologies for efficiently controlling arboviral diseases. The conference brought together 150 participants from 26 countries.

Implications of insecticide resistance for malaria vector control with long-lasting insecticidal nets: a WHO-coordinated, prospective, international, observational cohort study.

BACKGROUND: Scale-up of insecticide-based interventions has averted more than 500 million malaria cases since 2000. Increasing insecticide resistance could herald a rebound in disease and mortality. We aimed to investigate whether insecticide resistance was associated with loss of effectiveness of long-lasting insecticidal nets and increased malaria disease burden. METHODS: This WHO-coordinated, prospective, observational cohort study was done at 279 clusters (villages or groups of villages in which phenotypic resistance was measurable) in Benin, Cameroon, India, Kenya, and Sudan. Pyrethroid long-lasting insecticidal nets were the principal form of malaria vector control in all study areas; in Sudan this approach was supplemented by indoor residual spraying. Cohorts of children from randomly selected households in each cluster were recruited and followed up by community health workers to measure incidence of clinical malaria and prevalence of infection. Mosquitoes were assessed for susceptibility to pyrethroids using the standard WHO bioassay test. Country-specific results were combined using meta-analysis. FINDINGS: Between June 2, 2012, and Nov 4, 2016, 40 000 children were enrolled and assessed for clinical incidence during 1·4 million follow-up visits. 80 000 mosquitoes were assessed for insecticide resistance. Long-lasting insecticidal net users had lower infection prevalence (adjusted odds ratio [OR] 0·63, 95% CI 0·51-0·78) and disease incidence (adjusted rate ratio [RR] 0·62, 0·41-0·94) than did non-users across a range of resistance levels. We found no evidence of an association between insecticide resistance and infection prevalence (adjusted OR 0·86, 0·70-1·06) or incidence (adjusted RR 0·89, 0·72-1·10). Users of nets, although significantly better protected than non-users, were nevertheless subject to high malaria infection risk (ranging from an average incidence in net users of 0·023, [95% CI 0·016-0·033] per person-year in India, to 0·80 [0·65-0·97] per person year in Kenya; and an average infection prevalence in net users of 0·8% [0·5-1·3] in India to an average infection prevalence of 50·8% [43·4-58·2] in Benin). INTERPRETATION: Irrespective of resistance, populations in malaria endemic areas should continue to use long-lasting insecticidal nets to reduce their risk of infection. As nets provide only partial protection, the development of additional vector control tools should be prioritised to reduce the unacceptably high malaria burden. FUNDING: Bill & Melinda Gates Foundation, UK Medical Research Council, and UK Department for International Development.

A laboratory simulation study on suppression of resistance genes by differential exposures to an insecticide in Anopheles stephensi Liston population.

BACKGROUND & OBJECTIVES: : Insecticide applied at optimum dosage and coverage delays the development of resistance in disease vectors. The study was aimed to test the hypothesis whether decrease in exposure to insecticide leads to decrease in selection of insecticide resistance in mosquitoes. The mosquitoes were variably exposed to insecticide in the laboratory by simulating the variations in insecticide sprays applied in the field. METHODS: : The study was carried out on DDT resistant adults of Anopheles stephensi. Mosquitoes were differentially exposed to impregnated papers of DDT (4%), that were differentially masked to 25, 50, and 75% area with an unimpregnated Whatman No.1 filter paper, and to a positive control without any masking, i.e. 100% exposure area. The study was conducted for five generations and at each generation mosquitoes were exposed to differentially masked impregnated papers, and percent mortality was calculated. RESULTS: : The observed survival rate in differential exposures was more with the increase in heterozygous genotype resistance-susuceptible (RS) frequency. Resistant gene frequency with differential exposures (25 to 75%) was in the range of 0.38-0.54 for the F0 generation, which increased to 0.84-0.93 for the F4 generation. In 100% exposure it was 0.18 in F0 generation, which increased to 0.58 in the F4 generation. The resistant gene frequencies in the population showed increasing trend with decrease in exposure in contrast to complete exposure. INTERPRETATION & CONCLUSION: : Variable simulated exposures resulted in precipitation of increased resistance while complete exposure resulted in lower levels of resistance, signifying the importance of optimum dosage and coverage in the indoor residual spray in delaying/avoiding the development of insecticide resistance in the disease vectors.

Monitoring of long-lasting insecticidal nets (LLINs) coverage versus utilization: a community-based survey in malaria endemic villages of Central India.

BACKGROUND: Despite the known effectiveness of long-lasting insecticidal nets (LLINs) in providing protection against malaria, high level of ownership and use are very difficult to achieve and maintain. Nearly 40,000 LLINs were distributed in 2014 as an intervention tool against malaria transmission in 80 villages of Keshkal sub-district in Chhattisgarh, India. This study assessed LLIN coverage, access, utilization pattern, and key determinants for the net use 1 year after mass distribution. METHODS: In 2015, a cross-sectional household survey was carried out in 80 study clusters (whole village or part of village). From each cluster, 40 households were randomly selected and interviewed using a structured questionnaire adapted from the malaria indicator survey of Roll Back Malaria guidelines. Information on demographic characteristics, LLIN ownership, and its use on the night before the survey, and physical condition of LLINs were recorded. RESULTS: 2970 households were interviewed with a total of 15,003 individuals present in the households during the night before the survey. Nearly 98% of households had at least one LLIN and 59.4% of the surveyed population reportedly used an LLIN the previous night. LLIN use varied from 41 to 94% between the study clusters. Nearly 89% of the LLINs were found in good physical condition (without holes). However, proportion of household with at least one LLIN per two persons was only 39%. CONCLUSION: Universal coverage of LLINs was inadequate in the study clusters making it difficult for all household members to use an LLIN. LLIN use varied between clusters and was highest in children under 5 years of age. Health education campaigns and creating awareness about the benefit of sleeping under the LLINs in providing protection against malaria is required not only to high risk groups of pregnant women and children below 5 years of age but all the members of the family to have an epidemiological impact of this intervention at the community level. Relatively high net use despite poor access to LLINs indicates an overall desire to use nets when they are available. The main barrier to increased use of nets is the low coverage at household level.

Esterases are responsible for malathion resistance in Anopheles stephensi: A proof using biochemical and insecticide inhibition studies.

BACKGROUND & OBJECTIVES: Increase in prevalence and intensity of insecticide-resistance in vectors of vector-borne diseases is a major threat to sustainable disease control; and, for their effective management, studies on resistance mechanisms are important to come out with suitable strategies. Esterases are major class of detoxification enzymes in mosquitoes, which confers protection against insecticides in causing resistance. This study was aimed at biochemical characterization of esterases responsible for malathion resistance in Anopheles stephensi mosquitoes, along with its validation through biochemical techniques and native-PAGE assays. METHODS: Laboratory maintained susceptible and resistant An. stephensi mosquitoes were used for assessing the activity and effect of α - and ß -esterases on malathion. Bioassay, synergist bioassay, biochemical assay and native- PAGE were employed to characterize the role of esterases in conferring malathion-resistance. RESULTS: Notably significant (p < 0.0001) enhancement in α - and ß -esterases activity was observed with 2-fold increase in resistant An. StephensiGOA compared to susceptible An. StephensiBB. native-PAGE depicted two major bands 'a' (Rf = 0.80) and 'b' (Rf = 0.72) in susceptible An. stephensiBB , while one intense band 'b' (Rf = 0.72) was visible in resistant An. stephensiGOA. Inhibition assay revealed complete inhibition of α - and ß -esterases activity in presence of 1 mM malathion in susceptible strain compared to observed partial inhibition in resistant strain on native-PAGE. INTERPRETATION & CONCLUSION: This study provides a better understanding on the role of esterase enzyme (carboxylesterase) in conferring malathion-resistance in An. stephensi mosquitoes, as evident from the native-PAGE assay results. The study results could be used in characterizing the resistance mechanisms in vectors and for suggesting alternative chemical insecticide based resistance management strategies for effective vector-borne disease control.