Evaluation of the Long-Lasting Insecticidal Net Interceptor LN: Laboratory and Experimental Hut Studies against Anopheline and Culicine Mosquitoes in Northeastern Tanzania

Long lasting insecticidal nets (LN) are a primary method of malaria prevention. Before new types of LN are approved they need to meet quality and efficacy standards set by the WHO Pesticide Evaluation Scheme. The process of evaluation has three phases. In Phase I the candidate LN must meet threshold bioassay criteria after 20 standardized washes. In Phase II washed and unwashed LNs are evaluated in experimental huts against wild, free flying anopheline mosquitoes. In Phase III the LN are distributed to households in malaria endemic areas, sampled over three years of use and tested for continuing insecticidal efficacy. Interceptor® LN (BASF Corporation, Germany) is made of polyester netting coated with a wash resistant formulation of alpha-cypermethrin. Interceptor LN was subjected to bioassay evaluation and then to experimental hut trial against pyrethroid-susceptible Anopheles gambiae and An. funestus and resistant Culex quinquefasciatus. Mosquito mortality, blood feeding inhibition and personal protection were compared between untreated nets, conventional alpha-cypermethrin treated nets (CTN) washed 20 times and LNs washed 0, 20 and 30 times. In Phase I Interceptor LN demonstrated superior wash resistance and efficacy to the CTN. In the Phase II hut trial the LN killed 92% of female An. gambiae when unwashed and 76% when washed 20 times; the CTN washed 20 times killed 44%. The LN out-performed the CTN in personal protection and blood-feeding inhibition. The trend for An. funestus was similar to An. gambiae for all outcomes. Few pyrethroid-resistant Cx. quinquefasciatus were killed and yet the level of personal protection (75-90%) against Culex was similar to that of susceptible An. gambiae (76-80%) even after 20 washes. This protection is relevant because Cx. quinquefasciatus is a vector of lymphatic filariasis in East Africa. After 20 washes and 60 nights’ use the LN retained 27% of its initial insecticide dose. Interceptor LN meets the approval criteria set by WHO and is recommended for use in disease control against East African vectors of malaria and filariasis. Some constraints associated with the phase II evaluation criteria, in particular the washing procedure, are critically reviewed

Effectiveness of a long-lasting insecticide treatment kit (ICON® Maxx) for polyester nets over three years of household use: a WHO phase III trial in Tanzania.

BACKGROUND: ICON® Maxx (Syngenta) is an insecticide treatment kit of pyrethroid and binding agent for long-lasting treatment of mosquito nets. Interim recommendation for use on nets was granted by the World Health Organization (WHO) after successful evaluation in experimental huts following multiple washes. A full WHO recommendation is contingent upon demonstration of continued bio-efficacy after 3 years of use. METHODS: A household-randomized prospective study design was used to assess ICON Maxx-treated nets over 3 years in north-eastern Tanzania. Conventional treated nets (with lambda-cyhalothrin, but without binder) served as a positive control. At 6-monthly intervals, cross-sectional household surveys monitored net use and physical integrity, while cone and tunnel tests assessed insecticidal efficacy. Pyrethroid content was determined after 12 and 36 months. A parallel cohort of nets was monitored annually for evidence of net deterioration and attrition. RESULTS: After 12 months' use, 97% of ICON Maxx-treated nets but only 67% of CTN passed the WHO efficacy threshold for insecticidal durability (> 80% mortality in cone or tunnel or 90% feeding inhibition in tunnel). After 24- and 36-months use, 67% and 26% of ICON Maxx treated nets met the cone criteria, respectively, and over 90% met the combined cone and tunnel criteria. Lambda-cyhalothrin content after 36 months was 17% (15.8 ± 4.3 mg/m2) of initial content. ICON Maxx nets were used year-round and washed approximately 4 times per year. In cross-sectional survey after 36 months the average number of holes was 20 and hole index was 740 cm2 per net. Cohort nets had fewer holes and smaller hole index than cross-sectional nets. However, only 15% (40/264) of cohort nets were not lost to follow-up or not worn out after 36 months. CONCLUSIONS: Because more than 80% of nets met the WHO efficacy criteria after 36 months use, ICON Maxx was granted WHO full recommendation. Cross-sectional and cohort surveys were complementary and gave a fuller understanding of net durability. To improve net usage and retention, stronger incentives and health messaging should be introduced in WHO LLIN longitudinal trials. Untreated polyester nets may be made long-lastingly insecticidal in Africa through simple household treatment using ICON Maxx pyrethroid-binder kits

A new class of insecticide for malaria vector control: evaluation of mosquito nets treated singly with indoxacarb (oxadiazine) or with a pyrethroid mixture against Anopheles gambiae and Culex quinquefasciatus.

BACKGROUND: Universal coverage with long-lasting insecticidal mosquito nets (LLIN) or indoor residual spraying (IRS) of houses remain the primary strategies for the control of mosquito vectors of malaria. Pyrethroid resistant malaria vectors are widespread throughout sub-Saharan Africa and new insecticides with different modes of action are urgently needed if malaria vector control is to remain effective. Indoxacarb is an oxadiazine insecticide that is effective as an oral and contact insecticide against a broad spectrum of agricultural pests and, due to its unique site of action, no cross-resistance has been detected through mechanisms associated with resistance to insecticides currently used in public health. METHODS: WHO tunnel tests of host seeking mosquitoes were carried out as a forerunner to experimental hut trials, to provide information on dosage-dependent mortality, repellency, and blood-feeding inhibition. A dosage range of indoxacarb treated netting (100-1000 mg/m(2)) was tested against a pyrethroid susceptible strain of Anopheles gambiae. In addition, efficacy of indoxacarb 500 mg/m(2) was compared with a standard pyrethroid formulation against pyrethroid susceptible and resistant Culex quinquefasciatus. Dosages between 25 and 300 mg/m(2) indoxacarb were tested in tunnel tests and in ball-frame bioassays as mixtures with alphacypermethrin 25 mg/m(2) and were compared with singly applied treatments against an insectary reared pyrethroid resistant strain of Cx. quinquefasciatus originally collected in Cotonou, Benin. RESULTS: There was a dosage-dependent response in terms of indoxacarb induced mortality, with dosages >100 mg/m(2) producing the best mortality response. In tunnel tests indoxacarb 500 mg/m(2) exceeded WHOPES thresholds with >80 % mortality of adult An. gambiae and blood-feeding inhibition of 75 %. No cross-resistance to indoxacarb was detected through mechanisms associated with resistance to pyrethroid insecticides and was equally effective against susceptible and resistant strains of Cx. quinquefasciatus. Indoxacarb 500 mg/m(2) killed 75 % of pyrethroid resistant Cx. quinquefasciatus compared with only 21 % mortality with alphacypermethrin 40 mg/m(2). Mixtures of indoxacarb with pyrethroid produced an additive response for both mortality and blood-feeding inhibition. The best performing mixture (indoxacarb 200 mg/m(2) + alphacypermethrin 25 mg/m(2)) killed 83 % of pyrethroid resistant Cx. quinquefasciatus and reduced blood-feeding by 88 %, while alphacypermethrin only killed 36 % and inhibited blood-feeding by 50 %. CONCLUSIONS: New insecticides with different modes of action to those currently used in mosquito vector control are urgently needed. Indoxacarb shows great promise as a mixture with a pyrethroid and should be evaluated in experimental hut trials to determine performance against wild free-flying, pyrethroid resistant An. gambiae and wash-resistant formulations developed

Efficacy of interceptor® G2, a long-lasting insecticide mixture net treated with chlorfenapyr and alpha-cypermethrin against Anopheles funestus: experimental hut trials in north-eastern Tanzania.

BACKGROUND: The effectiveness of long-lasting insecticidal nets (LLIN), the primary method for preventing malaria in Africa, is compromised by evolution and spread of pyrethroid resistance. Further gains require new insecticides with novel modes of action. Chlorfenapyr is a pyrrole insecticide that disrupts mitochrondrial function and confers no cross-resistance to neurotoxic insecticides. Interceptor® G2 LN (IG2) is an insecticide-mixture LLIN, which combines wash-resistant formulations of chlorfenapyr and the pyrethroid alpha-cypermethrin. The objective was to determine IG2 efficacy under controlled household-like conditions for personal protection and control of wild, pyrethroid-resistant Anopheles funestus mosquitoes. METHODS: Experimental hut trials tested IG2 efficacy against two positive controls-a chlorfenapyr-treated net and a standard alpha-cypermethrin LLIN, Interceptor LN (IG1)-consistent with World Health Organization (WHO) evaluation guidelines. Mosquito mortality, blood-feeding inhibition, personal protection, repellency and insecticide-induced exiting were recorded after zero and 20 washing cycles. The trial was repeated and analysed using multivariate and meta-analysis. RESULTS: In the two trials held in NE Tanzania, An. funestus mortality was 2.27 (risk ratio 95% CI 1.13-4.56) times greater with unwashed Interceptor G2 than with unwashed Interceptor LN (p = 0.012). There was no significant loss in mortality with IG2 between 0 and 20 washes (1.04, 95% CI 0.83-1.30, p = 0.73). Comparison with chlorfenapyr treated net indicated that most mortality was induced by the chlorfenapyr component of IG2 (0.96, CI 0.74-1.23), while comparison with Interceptor LN indicated blood-feeding was inhibited by the pyrethroid component of IG2 (IG2: 0.70, CI 0.44-1.11 vs IG1: 0.61, CI 0.39-0.97). Both insecticide components contributed to exiting from the huts but the contributions were heterogeneous between trials (heterogeneity Q = 36, P = 0.02). WHO susceptibility tests with pyrethroid papers recorded 44% survival in An. funestus. CONCLUSIONS: The high mortality recorded by IG2 against pyrethroid-resistant An. funestus provides first field evidence of high efficacy against this primary, anthropophilic, malaria vector

Bio-efficacy and wash-fastness of a lambda-cyhalothrin long-lasting insecticide treatment kit (ICON® Maxx) against mosquitoes on various polymer materials.

BACKGROUND: Long-lasting efficacy of insecticide-treated nets is a balance between adhesion, retention and migration of insecticide to the surface of netting fibres. ICON® Maxx is a twin-sachet 'home-treatment kit' of pyrethroid plus binding agent, recommended by the World Health Organization (WHO) for long-lasting, wash-fast treatment of polyester nets. While knitted polyester netting is widely used, fine woven polyethylene netting is increasingly available and nets made of cotton and nylon are common in Africa and Asia. It is important to investigate whether ICON Maxx is able to fulfill the WHO criteria of long-lasting treatment on a range of domestic fabrics to widen the scope for malaria protection. METHODS: This study was a controlled comparison of the bio-efficacy and wash-fastness of lambda-cyhalothrin CS, with or without binder, on nets made of cotton, polyethylene, nylon, dyed and undyed polyester. Evaluation compared an array of bioassays: WHO cone and cylinder, median time to knockdown and WHO tunnel tests using Anopheles mosquitoes. Chemical assay revealed further insight. RESULTS: ICON Maxx treated polyethylene and polyester netting met the WHO cone and tunnel test bio-efficacy criteria for LLIN after 20 standardized washes. Although nylon and cotton netting failed to meet the WHO cone and cylinder criteria, both materials passed the WHO tunnel test criterion of 80% mortality after 20 washes. All materials treated with standard lambda-cyhalothrin CS without binder failed to meet any of the WHO bio-efficacy criteria within 5 washes. CONCLUSION: The bio-efficacy of ICON Maxx against mosquitoes on netting washed up to 20 times demonstrated wash durability on a range of synthetic polymer and natural fibres: polyester, polyethylene, nylon and cotton. This raises the prospect of making insecticide-binder kits into an effective approach for turning untreated nets, curtains, military clothing, blankets-and tents and tarpaulins as used in disasters and humanitarian emergencies-into effective malaria prevention products. It may provide a solution to the problem of reduced LLIN coverage between campaigns by converting commercially sourced untreated nets into LLINs through community or home treatment. It may also open the door to binding of non-pyrethroid insecticides to nets and textiles for control of pyrethroid resistant vectors

Evaluation of ICON Maxx, a long-lasting treatment kit for mosquito nets: experimental hut trials against anopheline mosquitoes in Tanzania.

BACKGROUND: Insecticide-treated nets are the primary method of preventing malaria. To remain effective, the pyrethroid insecticide must withstand multiple washes over the lifetime of the net. ICON(®) Maxx is a 'dip-it-yourself' kit for long-lasting treatment of polyester nets. The twin-sachet kit contains a slow-release capsule suspension of lambda-cyhalothrin plus binding agent. To determine whether ICON Maxx meets the standards required by the World Health Organization Pesticide Evaluation Scheme (WHOPES), the efficacy and wash fastness of ICON Maxx was evaluated against wild, free-flying anopheline mosquitoes. METHODS: ICON Maxx was subjected to bioassay evaluation and experimental hut trial against pyrethroid-susceptible Anopheles gambiae, Anopheles arabiensis and Anopheles funestus. Mosquito mortality, blood feeding inhibition and personal protection were compared between untreated nets, conventional lambda-cyhalothrin treated nets (CTN) washed either four times (cut-off threshold) or 20 times, and ICON Maxx-treated nets either unwashed or washed 20 times. RESULTS: In bioassay, ICON Maxx demonstrated superior wash resistance to the CTN. In the experimental hut trial, ICON Maxx killed 75 % of An. funestus, 71 % of An. gambiae and 47 % of An. arabiensis when unwashed and 58, 66 and 42 %, respectively, when 20 times washed. The CTN killed 52 % of An. funestus, 33 % of An. gambiae and 30 % of An. arabiensis when washed to the cut-off threshold of four washes and 40, 40 and 36 %, respectively, when 20 times washed. Percentage mortality with ICON Maxx 20 times washed was similar (An. funestus) or significantly higher (An. gambiae, An. arabiensis) than with CTN washed to the WHOPES cut-off threshold. Blood-feeding inhibition with ICON Maxx 20 times washed was similar to the CTN washed to cut-off for all three species. Personal protection was significantly higher with ICON Maxx 20 times washed (66-79 %) than with CTN washed to cut-off (48-60 %). CONCLUSIONS: Nets treated with ICON Maxx and washed 20 times met the approval criteria set by WHOPES for Phase II trials in terms of mortality and blood-feeding inhibition. This finding raises the prospect of conventional polyester nets and other materials being made long-lastingly insecticidal through simple dipping in community or home, and thus represents a major advance over conventional pyrethroid treatments

An experimental hut evaluation of Olyset® nets against anopheline mosquitoes after seven years use in Tanzanian villages

BACKGROUND: Long-lasting insecticidal nets (LLINs) are advocated by WHO for protection against malaria. Of the three brands of LLINs currently approved by WHO, Olyset(R) is the only one currently granted full recommendation. With this type of LLIN, the insecticide (permethrin) is incorporated into the polyethylene fibre during manufacture and diffuses from the core to the surface, thereby maintaining surface concentrations. It has not been determined for how long Olyset nets remain protective against mosquitoes in household use. METHODS: Examples of Olyset nets, which had been in use in Tanzanian villages for seven years, were tested in experimental huts against naturally entering Anopheles gambiae and Anopheles funestus mosquitoes. Performance was compared with new Olyset nets, conventionally treated ITNs (either newly treated with alphacypermethrin or taken from local villages after 1.5 years of use) and untreated nets. All nets were artificially holed except for the seven-year Olyset nets, which had developed holes during prolonged domestic use. RESULTS: Anopheles funestus and An. gambiae in NE Tanzania are susceptible to pyrethroids. The new Olyset nets caused high mortality against An. funestus (73.9%) and An. gambiae (62.7%) in experimental huts. The seven-year Olyset nets caused 58.9% mortality against An. funestus and 40.0% mortality against An. gambiae. The freshly treated alphacypermethrin nets also caused high mortality against An. funestus (70.6%) and An. gambiae (72.0%); this decreased to 58.4% and 69.6% respectively after 1.5 years of use. The new Olyset nets inhibited blood-feeding by 40-50%. The 7 year Olyset nets showed no feeding inhibition over that shown by the untreated nets. The alphacypermethrin treated nets failed to inhibit blood-feeding after 1.5 years of use. However iHhhdn laboratory tunnel tests samples of all types of treated net including the 7 year Olyset inhibited blood-feeding by more than 95%. CONCLUSION: After seven years of use Olyset nets were still strongly insecticidal. Mosquito mortality decreased by only 20-35% over this period. However, Olyset would not provide personal protection after seven years unless it was in good condition and all holes fully repaired

Preferred resting surfaces of dominant malaria vectors inside different house types in rural south-eastern Tanzania

Background: Malaria control in Africa relies extensively on indoor residual spraying (IRS) and insecticide-treated nets (ITNs). IRS typically targets mosquitoes resting on walls, and in few cases, roofs and ceilings, using contact insecticides. Unfortunately, little attention is paid to where malaria vectors actually rest indoors, and how such knowledge could be used to improve IRS. This study investigated preferred resting surfaces of two major malaria vectors, Anopheles funestus and Anopheles arabiensis, inside four common house types in rural south-eastern Tanzania. Methods: The assessment was done inside 80 houses including: 20 with thatched roofs and mud walls, 20 with thatched roofs and un-plastered brick walls, 20 with metal roofs and un-plastered brick walls, and 20 with metal roofs and plastered brick walls, across four villages. In each house, resting mosquitoes were sampled in mornings (6 a.m.–8 a.m.), evenings (6 p.m.–8 p.m.) and at night (11 p.m.–12.00 a.m.) using Prokopack aspirators from multiple surfaces (walls, undersides of roofs, floors, furniture, utensils, clothing, curtains and bed nets). Results: Overall, only 26% of An. funestus and 18% of An. arabiensis were found on walls. In grass-thatched houses, 33–55% of An. funestus and 43–50% of An. arabiensis rested under roofs, while in metal-roofed houses, only 16–20% of An. funestus and 8–30% of An. arabiensis rested under roofs. Considering all data together, approximately 40% of mosquitoes rested on surfaces not typically targeted by IRS, i.e. floors, furniture, utensils, clothing and bed nets. These proportions were particularly high in metal-roofed houses (47–53% of An. funestus; 60–66% of An. arabiensis). Conclusion: While IRS typically uses contact insecticides to target adult mosquitoes on walls, and occasionally roofs and ceilings, significant proportions of vectors rest on surfaces not usually sprayed. This gap exceeds one-third of malaria mosquitoes in grass-thatched houses, and can reach two-thirds in metal-roofed houses. Where field operations exclude roofs during IRS, the gaps can be much greater. In conclusion, there is need for locally-obtained data on mosquito resting behaviours and how these influence the overall impact and costs of IRS. This study also emphasizes the need for alternative approaches, e.g. house screening, which broadly tackle mosquitoes beyond areas reachable by IRS and ITNs

Assessing the variability in experimental hut trials evaluating insecticide-treated nets against malaria vectors.

Experimental hut trials (EHTs) are used to evaluate indoor vector control interventions against malaria vectors in a controlled setting. The level of variability present in the assay will influence whether a given study is well powered to answer the research question being considered. We utilised disaggregated data from 15 previous EHTs to gain insight into the behaviour typically observed. Using simulations from generalised linear mixed models to obtain power estimates for EHTs, we show how factors such as the number of mosquitoes entering the huts each night and the magnitude of included random effects can influence study power. A wide variation in behaviour is observed in both the mean number of mosquitoes collected per hut per night (ranging from 1.6 to 32.5) and overdispersion in mosquito mortality. This variability in mortality is substantially greater than would be expected by chance and should be included in all statistical analyses to prevent false precision of results. We utilise both superiority and non-inferiority trials to illustrate our methodology, using mosquito mortality as the outcome of interest. The framework allows the measurement error of the assay to be reliably assessed and enables the identification of outlier results which could warrant further investigation. EHTs are increasingly playing an important role in the evaluation and regulation of indoor vector control interventions so it is important to ensure that these studies are adequately powered. [Abstract copyright: © 2023 The Authors.

Systematic review of the entomological impact of insecticide-treated nets evaluated using experimental hut trials in Africa.

Resistance of anopheline mosquitoes to pyrethroid insecticides is spreading rapidly across sub-Saharan Africa, diminishing the efficacy of insecticide-treated nets (ITNs) - the primary tool for preventing malaria. The entomological efficacy of indoor vector control interventions can be measured in experimental hut trials (EHTs), where hut structures resemble local housing, but allow the collection of mosquitoes that entered, exited, blood-fed and/or died. There is a need to understand how the spread of resistance changes ITN efficacy and to elucidate factors influencing EHT results, including differences in experimental hut design, to support the development of novel vector control tools. A comprehensive database of EHTs was compiled following a systematic review to identify all known trials investigating ITNs or indoor residual spraying across sub-Saharan Africa. This analysis focuses on EHTs investigating ITNs and uses Bayesian statistical models to characterise the complex interaction between ITNs and mosquitoes, the between-study variability, and the impact of pyrethroid resistance. As resistance rises, the entomological efficacy of ITNs declines. They induce less mortality and are less likely to deter mosquitoes from entering huts. Despite this, ITNs continue to offer considerable personal protection by reducing mosquito feeding until resistance reaches high levels. There are clear associations between the different entomological impacts of ITNs, though there is still substantial variability between studies, some of which can be accounted for by hut design. The relationship between EHT outcomes and the level of resistance (as measured by discriminating dose bioassays) is highly uncertain. The meta-analyses show that EHTs are an important reproducible assay for capturing the complex entomological efficacy of ITNs on blood-feeding mosquitoes. The impact of pyrethroid resistance on these measures appears broadly consistent across a wide geographical area once hut design is accounted for, suggesting results can be extrapolated beyond the sites where the trials were conducted. Further work is needed to understand factors influencing EHT outcomes and how the relationship between outcomes and resistance varies when different methods are used to assess the level of resistance in wild mosquito populations. This will allow more precise estimates of the efficacy of these important vector control tools